Anti-her2 antibody variants

ABSTRACT

The present invention concerns novel antibody variants, particularly anti-HER2 antibody variants having substitutions at positions within the variable domains of the heavy and light chains

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/237,009, filed Sep. 24, 2008, which is a continuation of U.S.application Ser. No. 10/410,894, filed Apr. 9, 2003, which claims thebenefit under 35 U.S.C. 119(h) of provisional application Ser. No.60/371,609, filed Apr. 10, 2002, which are incorporated by referenceherein in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention concerns novel antibody variants, particularlyanti-HER2 antibody variants.

Description of the Related Art

Members of the ErbB family of receptor tyrosine kinases are importantmediators of cell growth, differentiation and survival. The receptorfamily includes four distinct members, including epidermal growth factorreceptor (EGFR or ErbB1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) andHER4 (ErbB4 or tyro2).

p185^(neu), was originally identified as the product of the transforminggene from neuroblastomas of chemically treated rats. The activated formof the neu proto-oncogene results from a point mutation (valine toglutamic acid) in the transmembrane region of the encoded protein.Amplification of the human homolog of neu is observed in breast andovarian cancers and correlates with a poor prognosis (Slamon et al.,Science, 235:177-182 (1987); Slamon et al., Science 244(4905):707-12(1989); and U.S. Pat. No. 4,968,603). To date, no point mutationanalogous to that in the neu proto-oncogene has been reported for humantumors. Overexpression of ErbB2 (frequently but not uniformly due togene amplification) has also been observed in other carcinomas includingcarcinomas of the stomach, endometrium, salivary gland, lung, kidney,colon, thyroid, pancreas and bladder. See, among others, King et al.,Science, 229:974 (1985); Yokota et al., Lancet, 1:765-767 (1986);Fukushigi et al., Mol Cell Biol., 6:955-958 (1986); Gcurin et al.,Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989);Yoncmura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol.Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990);Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res.,49:6605 (1989); Zhau et al., Mol. Carcinog., 3:354-357 (1990); Aaslandet al. Br. J. Cancer, 57:358-363 (1988); Williams et al. Pathobiology,59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). ErbB2 maybe overexpressed in prostate cancer (Gu et al. Cancer Lett., 99:185-9(1996); Ross et al. Hum. Pathol., 28:827-33 (1997); Ross et al. Cancer,79:2162-70 (1997); and Sadasivan et al. J. Urol., 150:126-31 (1993)).

Antibodies directed against the rat p185^(neu) and human ErbB2 proteinproducts have been described. Drebin and colleagues have raisedantibodies against the rat neu gene product, p185^(neu). See, forexample, Drebin et al., Cell, 41:695-706 (1985); Myers et al., Meth.Enzym. 198:277-290 (1991); and WO94/22478. Drebin et al. Oncogene2:273-277 (1988) report that mixtures of antibodies reactive with twodistinct regions of p185^(neu) result in synergistic anti-tumor effectson neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S.Pat. No. 5,824,311 issued Oct. 20, 1998.

Other anti-ErbB2 antibodies with various properties have been describedin Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancocket al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994);Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

Hudziak et al., Mol Cell Biol 9(3): 1165-72 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SK-BR-3. Relative cellproliferation of the SK-BR-3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel reduced cellular proliferation to a lesserextent in this assay. The antibody 4D5 was further found to sensitizeErbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNF-α. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. Theanti-ErbB2 antibodies discussed in Hudziak et al., A., Mol Cell Biol9(3):1165-72 (1989) are further characterized in Fendly et al., CancerRes 50(5):1550-8 (1990); Kotts et al. In Vitro 26(3):59A (1990); Sarupet al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin.Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol.11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother.37:255-263 (1993); Pictras et al. Oncogene 9:1829-1838 (1994); Vitettaet al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol.Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem.266:14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206(1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaeferet al. Oncogene 15:1385-1394 (1997).

The murine monoclonal anti-HER2 antibody inhibits the growth of breastcancer cell lines that overexpress HER2 at the 2+ and 3+ level, but hasno activity on cells that express lower levels of HER2 (Lewis et al.,Cancer Immunol. Immunother. [1993]). Based on this observation, antibody4D5 was humanized (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 [1992]). The humanized version designated HERCEPTIN®(huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337) was tested in breastcancer patients whose tumors overexpress HER2 but who had progressedafter conventional chemotherapy (Cobleigh et al., J. Clin. Oncol. 17:2639-2648 [1999]). Most patients in this trial expressed HER2 at the 3+level, though a fraction was 2+ tumors. Remarkably. HERCEPTIN® inducedclinical responses in 15% of patients (complete responses in 4% ofpatients, and partial responses in 11%) and the median duration of thoseresponses was 9.1 months. HERCEPTIN® received marketing approval fromthe Food and Drug Administration Sep. 25, 1998 for the treatment ofpatients with metastatic breast cancer whose tumors overexpress theErbB2 protein.

SUMMARY OF THE INVENTION

The present invention is based on the finding that particular aminoacids of the humanized anti-HER2 antibody hu4D5-8, determined by alaninescanning to be necessary for antigen binding, and other amino acidsfound by alanine scanning to be relatively unimportant for antigenbinding, may be substituted to produce variants having high affinity forHER2. Preferred positions for possible mutations are shown in FIG. 2.Thus, while certain variants are discussed in detail herein, othervariants with substitutions at one or more of the positions indicated inFIG. 2 are also contemplated and encompassed by the present invention.

In one aspect, the present invention relates to a polypeptide whichcomprises an antibody light chain variable domain comprising thehypervariable regions of SEQ ID NO: 1 wherein one or more amino acidsselected from the group consisting of Q27(V_(L)), D28(V_(L)),N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)), Y55(V_(L)),R66(V_(L)), H91(V_(L)), Y92(V_(L)), and T94(V_(L)), numbered accordingto the Kabat numbering system, are substituted with any amino acid otherthan alanine.

In one embodiment, the invention relates to a polypeptide wherein thehypervariable regions of SEQ ID NO: 1 comprise amino acid substitutionsat one or more positions selected from the group consisting ofN30(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L), Y92(V_(L)), andT94(V_(L)), and F100(V_(L)).

In another embodiment, the invention concerns a polypeptide wherein thehypervariable regions of SEQ ID NO: 1 comprise amino acid substitutionsat one or more positions selected from the group consisting ofN30(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L)), Y92(V_(L)), andT94(V_(L)).

In yet another embodiment, the invention concerns a polypeptide whereinthe hypervariable regions of SEQ ID NO: 1 comprise one or more aminoacid substitutions selected from the group consisting of D28(V_(L))Q;D28(V_(L))G; N30(V_(L))S; T31(V_(L))S; A32(V_(L))G; Y49(V_(L))W,Y49(V_(L))D, Y49(V_(L))V; F53(V_(L))W. F53(V_(L))V, F53(V_(L))Q,Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F, H91(V_(L))Y, Y92(V_(L))W, andT94(V_(L))S.

In a further embodiment, the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of D28(V_(L))Q; D28(V_(L))G; N30(V_(L))S; T31(V_(L))S;A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D, Y49(V_(L))V; F53(V_(L))W,F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F,H91(V_(L))Y, and Y92(V_(L))W

In a still further embodiment, the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of Y49(V_(L))D, F53(V_(L))W, and Y55(V_(L))W. In specificembodiments, the polypeptide can contain two or three of the indicatedamino acid substitutions.

In a further embodiment, the invention concerns a polypeptide, whereinthe hypervariable regions of SEQ ID NO: 1 comprise one or more aminoacid substitutions selected from the group consisting of N30(V_(L))S,F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F, Y92(V_(L))W and T94(V_(L))S. In aspecific embodiment, N30(V_(L)) is substituted with S, H91(V_(L)) issubstituted with F, and Y92(V_(L)) is substituted with W.

In all embodiments, the polypeptide can, for example, be an antibody,such as a humanized (including chimeric) or human antibody, includingantibody fragments, such as, for example, Fv, Fab, Fab′ and F(ab′)₂fragments.

In another aspect, the invention concerns a polypeptide which comprisesan antibody heavy chain variable domain comprising the hypervariableregions of SEQ ID NO: 2 wherein one or more amino acids selected fromthe group consisting of W95(V_(H)), D98(V_(H)), F100(V_(H)),Y100a(V_(H)), and Y102(V_(H)), numbered according to the Kabat numberingsystem, are substituted with any amino acid other than alanine.

In one embodiment, in the foregoing polypeptide, the hypervariableregions of SEQ ID NO: 2 comprise one or more amino acid substitutionsselected from the group consisting of W95(V_(H))Y, D98(V_(H))W,D98(V_(H))R, D98(V_(H))K, D98(V_(H))H, F100(V_(H))P, Y100a(V_(H))F,Y102(V_(H))V, Y102(V_(H))K, and Y102(V_(H))L.

In another embodiment, the hypervariable regions of SEQ ID NO: 2comprise one or more amino acid substitutions selected from the groupconsisting of, D98(V_(H))W, Y100a(V_(H))F, and Y102(V_(H))V.

In yet another embodiment, the hypervariable regions of SEQ ID NO: 2comprise one or more amino acid substitutions selected from the groupconsisting of F100(V_(H))P and Y102(V_(H))K.

In a specific embodiment, the polypeptide comprises the amino acidsubstitutions F100(V_(H))P and Y102(V_(H))K.

Just as before, the polypeptide can, for example be an antibody, such asa humanized (including chimeric), or human antibody, including antibodyfragments, such as, e.g. Fv, Fab, Fab′ and F(ab′)₂ fragments.

In a further aspect, the invention concerns an antibody that is capableof binding to the extracellular domain of HER2, which comprises thehypervariable regions of SEQ ID NO: 1 wherein one or more amino acidsselected from the group consisting of Q27(V_(L)), D28(V_(L)),N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)), Y55(V_(L)),R66(V_(L)), H91(V_(L)), Y92(V_(L)), and T94(V_(L)), numbered accordingto the Kabat numbering system, are substituted with any amino acid otherthan alanine.

In one embodiment, in the antibody one or more amino acids selected fromthe group consisting of N30(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L)),Y92(V_(L)), and T94(V_(L)), numbered according to the Kabat numberingsystem, are substituted with any amino acid other than alanine.

In another embodiment, one or more amino acids selected from the groupconsisting of N30(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L)),Y92(V_(L)), and T94(V_(L)), numbered according to the Kabat numberingsystem, are substituted with any amino acid other than alaninc.

In yet another embodiment, the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of D28(V_(L))Q; D28(V_(L))G; N30(V_(L))S; T31(V_(L))S;A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D, Y49(V_(L))V; F53(V_(L))W,F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F,H91(V_(L))Y, Y92(V_(L))W, and T94(V_(L))S.

In a further embodiment, the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of D28(V_(L))Q; D28(V_(L))G; N30(V_(L))S; T31(V_(L))S;A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D, Y49(V_(L))V; F53(V_(L))W,F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F,H91(V_(L))Y, and Y92(V_(L))W.

In a still further embodiment, the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of Y49(V_(L))D, F53(V_(L))W, and Y55(V_(L))W. In specificembodiments, the antibody may contain two or three, e.g. all three, ofthe indicated substitutions.

The invention specifically includes antibodies in which thehypervariable regions of SEQ ID NO: 1 comprise one or more amino acidsubstitutions selected from the group consisting of N30(V_(L))S,F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F, Y92(V_(L))W, T94(V_(L))S. Thus,in a particular embodiment, the invention concerns an antibody whereinN30(V_(L)) is substituted with S, H91(V_(L)) is substituted with F, andY92(V_(L)) is substituted with W.

The antibodies include humanized (including chimeric) and humanantibodies, including antibody fragments, e.g. Fv, Fab, Fab′ and F(ab′)₂fragments.

In a further aspect, the invention concerns an antibody that is capableof binding to the extracellular domain of HER2, which comprises anantibody heavy chain variable domain comprising the hypervariableregions of SEQ ID NO: 2 wherein one or more amino acids selected fromthe group consisting of W95(V_(H)), D98(V_(H)), F100(V_(H)),Y100a(V_(H)), and Y102(V_(H)), numbered according to the Kabat numberingsystem, are substituted with any amino acid other than alanine.

In one embodiment, the invention concerns an antibody wherein thehypervariable regions of SEQ ID NO: 2 comprise one or more amino acidsubstitutions selected from the group consisting of W95(V_(H))Y,D98(V_(H))W, D98(V_(H))R, D98(V_(H))K, D98(V_(H))H, F100(V_(H))P,F100(V_(H))L, F100(V_(H))M, Y100a(V_(H))F, Y102(V_(H))V, Y102(V_(H))K,and Y102(V_(H))L.

In another embodiment, in the antibody of the present invention thehypervariable regions of SEQ ID NO: 2 comprise one or more amino acidsubstitutions selected from the group consisting of, D98(V_(H))W,Y100a(V_(H))F, and Y102(V_(H))V.

In yet another embodiment, the hypervariable regions of SEQ ID NO: 2comprise one or more amino acid substitutions selected from the groupconsisting of F100(V_(H))P and Y102(V_(H))K or Y102(V_(H))L. Theantibody may, for example, comprise the amino acid substitutionsF100(V_(H))P and Y102(V_(H))K, or F100(V_(H))P and Y102(V_(H))L.

Just as in other aspects of the invention, the antibody can be humanized(including chimeric), or human, including antibody fragments, such as,for example, Fv, Fab, Fab′ and F(ab′)₂ fragments.

In a further aspect, the invention concerns an antibody that is capableof binding to the extracellular domain of HER2, which comprises thehypervariable regions of SEQ ID NOs: 1 and 2 wherein one or more aminoacids selected from the group consisting of Q27(V_(L)), D28(V_(L)),N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)), Y55(V_(L)),R66(V_(L)), H91(V_(L)), Y92(V_(L)), T94(V_(L)), W95(V_(H)), D98(V_(H)),F100(V_(H)), Y100a(V_(H)), and Y102(V_(H)), numbered according to theKabat numbering system, are substituted with any amino acid other thanalanine.

In one embodiment, in the entibody the hypervariable regions of SEQ IDNOs: 1 and 2 comprise one or more amino acid substitutions selected fromthe group consisting of N30(V_(L))S, F53(V_(L))W, Y55(V_(L))W,H91(V_(L))F, Y92(V_(L))W, T94(V_(L))S, D98(V_(H))W, Y100a(V_(H))F, andY102(V_(H))V.

In another embodiment, N30(V_(L)) is substituted with S, H91(V_(L)) issubstituted with F, Y92(V_(L)) is substituted with W, T94(V_(L)) issubstituted with S, D98(V_(H)) is substituted with W, Y100a(V_(H)) issubstituted with F, and Y102(V_(H)) is substituted with V.

In a specific embodiment, D98(V_(H)) is substituted with W.

In yet another embodiment, the hypervariable regions of SEQ ID NOs 1 and2 comprise one or more amino acid substitutions selected from the groupconsisting of D28(V_(L))Q; D28(V_(L))G; N30(V_(L))S; T31(V_(L))S;A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D, Y49(V_(L))V; F53(V_(L))W,F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F,H91(V_(L))Y, Y92(V_(L))W, T94(V_(L))S, F100(V_(L))W; W95(V_(H))Y,D98(V_(H))W, D98(V_(H))R, D98(V_(H))K, D98(V_(H))H, F100(V_(H))P,F100(V_(H))L, F100(V_(H))M, Y100a(V_(H))F, Y102(V_(H))V, Y102(V_(H))K,and Y102(V_(H))L.

In a further embodiment, the hypervariable regions of SEQ ID NOs 1 and 2comprise one or more amino acid substitutions selected from the groupconsisting of Y49(V_(L))D, F53(V_(L))W, Y55(V_(L))W, D98(V_(H))W,F100(V_(H))P, and Y102(V_(H))L.

In a still further embodiment, the hypervariable regions of SEQ ID NOs:1 and 2 comprise the following substitutions: Y49(V_(L))D, F53(V_(L))W,Y55(V_(L))W, F100(V_(H))P, and Y102(V_(H))L. In a particular embodiment,the hypervariable regions of SEQ ID NO 2 may further comprise thesubstitution D98(V_(H))W.

In an additional embodiment, the binding affinity of the antibody forthe HER2 extracellular domain is at least about three-fold better thanthe binding affinity of humanized monoclonal antibody 4D5-8 for the HER2extracellular domain.

Again, the antibody can, for example, be humanized (including chimeric),or human, including antibody fragments, such as Fv, Fab, Fab′ andF(ab′)₂ fragments.

In a different aspect, the invention concerns an antibody that iscapable of binding to the extracellular domain of HER2 which comprisesthe light chain variable domain of SEQ ID NO: 1 wherein one or moreamino acids selected from the group consisting of Q27(V_(L)),N30(V_(L)), Y49(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L)), Y92(V_(L)),and T94(V_(L)), numbered according to the Kabat numbering system, aresubstituted with any amino acid other than alanine.

In a particular embodiment, the light chain variable domain of SEQ IDNO: 1 comprises one or more amino acid substitutions selected from thegroup consisting of N30(V_(L))S, Y49(V_(L))F, Y49(V_(L))W, F53(V_(L))W,Y55(V_(L))W, H91(V_(L))F, Y92(V_(L))W, and T94(V_(L))S.

In another embodiment, N30(V_(L)) is substituted with S, H91(V_(L)) issubstituted with F, and Y92(V_(L)) is substituted with W.

In a still further aspect, the invention concerns a humanized anti-HER2antibody 4D5-8, comprising one or more amino acid substitutions selectedfrom the group consisting of N30(V_(L))S, Y49(V_(L))F, Y49(V_(L))W,F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F, Y92(V_(L))W, T94(V_(L))S,D98(V_(H))W, F100(V_(H))P, Y100a(V_(H))F, and Y102(V_(H))V, numberedaccording to the Kabat numbering system.

In one embodiment, the humanized anti-HER2 antibody 4D5-8 comprises oneor more amino acid substitutions selected from the group consisting ofY49(V_(L)D, F53(V_(L))W, Y55(V_(L))W, F100(V_(H))P, Y102(V_(H))K, andY102(V_(H))L.

In another embodiment, the humanized anti-HER2 antibody 4D5-8 furthercomprises the amino acid substitution D98(V_(H))W.

In yet another embodiment, the humanized anti-HER2 antibody 4D5-8comprises one or more of the amino acid substitutions selected from thegroup consisting of Y49(V_(L))D, F53(V_(L))W, and Y55(V_(L))W.

In a further embodiment, the humanized anti-HER2 antibody 4D5-8comprises one or more of the amino acid substitutions selected from thegroup consisting of F100(V_(H))P, Y102(V_(H))K, and Y102(V_(H))L.

In a still further embodiment, the humanized anti-HER2 antibody 4D5-8comprises the following amino acid substitutions: Y49(V_(L))D,F53(V_(L))W, Y55(V_(L))W, F100(V_(H))P, and Y102(V_(H))K.

In a different embodiment, the humanized anti-HER2 antibody 4D5-8comprises the following amino acid substitutions: Y49(V_(L))D,F53(V_(L))W, Y55(V_(L))W, F100(V_(H))P, and Y102(V_(H))L.

In another aspect, the invention concerns an article of manufacturecomprising a container, a composition contained therein, and a packageinsert or label indicating that the composition can be used to treatcancer characterized by the overexpression of HER2, wherein thecomposition comprises the antibody of Claim 60 or 61. The cancer may,for example, be breast cancer.

In yet another aspect, the invention concerns an antibody variant of aparent antibody which binds HER2, comprising an amino acid substitutionat position 98 of a heavy chain variable domain thereof, and wherein thebinding affinity of the antibody variant for HER2 is better than thebinding affinity of the parent antibody for HER2. In a specificembodiment, the amino acid at position 98 is substituted with W. Theparent antibody may be, without limitation, a humanized antibody.

In a different aspect, the invention concerns a method for isolatinghigh-affinity variants of a humanized anti-HER2 antibody, comprising:

(a) producing anti-HER2 variants with substitutions at one or more aminoacids selected from the group consisting Q27(V_(L)), D28(V_(L)),N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)), Y55(V_(L)),R66(V_(L)), H91(V_(L)), Y92(V_(L)), T94(V_(L)), W95(V_(H)), D98(V_(H)),F100(V_(H)), Y100a(V_(H)), and Y102(V_(H)), within the hypervariableregions of SEQ ID NOs: 1 and 2, wherein numbering is according to theKabat numbering system;

(b) measuring binding affinities of the variants produced in (a) forHER2 extracellular domain; and

(c) selecting for high-affinity variants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the light chain variable domain (V_(L)) amino acidresidues of huMAb4D5-8 (SEQ ID NO. 1). FIG. 1B shows the heavy chainvariable domain (V_(H)) amino acid residues of huMAb4D5-8 (SEQ ID NO.2). Both FIGS. 1A and 1B use the generally accepted numbering schemefrom Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987)). In FIGS.1A and 1B the hypervariable region residues are identified byunderlining. As discussed in more detail below, the hypervariableregions were determined according to both a standard sequence definition(Kabat, E. A. et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987)) and a structuraldefinition (Chothia, C. & Lesk, A. M., J Mol Biol 196(4):901-17 (1987)).In FIG. 1A, these regions are designated as V_(L)-hypervariable region 1(comprising the amino acid sequence RASQDVNTAVA (SEQ ID NO: 19)),V_(L)-hypervariable region 2 (comprising the amino acid sequence SASFLYS(SEQ ID NO: 20)), and V_(L)-hypervariable region 3 (comprising the aminoacid sequence QQHYTTPPT (SEQ ID NO: 21)). In FIG. 1B, the hypervariableregions are designated as V_(H)-hypervariable region 1 (comprising theamino acid sequence GFNIKDTYIH (SEQ ID NO: 22)), V_(H)-hypervariableregion 2 (comprising the amino acid sequence RIYPTNGYTRYADSVKG (SEQ IDNO: 23)), and V_(H)-hypervariable region 3 (comprising the amino acidsequence WGGDGFYAMDY (SEQ ID NO: 24)).

FIG. 2A shows the residue positions mutated in phage displayedlibraries. Nineteen residues on the surface of the hu4D5-8 Fab werefully randomized using NNS codon degeneracy. The randomized residues(Kabat numbering; Johnson, G. & Wu, T. T., Nucleic Acids Res 29(1):205-6(2001)) were grouped by their location on the surface of the antibodystructure into five libraries, shown in FIG. 2B. Some residues wereincluded in more than one library to test for context-dependent effects.

FIG. 3 shows amino acid substitutions in Fab-phage clones. For eachposition, the source library, wild-type residue, and the most commonlyobserved residue (ϕ) after 4 rounds of selection are shown. The observedfrequency (%) of each amino acid at each position (Kabat numbering;Johnson, G. & Wu, T. T., Nucleic Acids Res 29(1):205-6 (2001)) wascalculated based upon the number of unique sequenced clones (N_(G); thatis, removing sibling clones) and normalized for degenerate codons, codonbias and the total number of unique sequences (siblings removed). Thewild-type and most-common frequencies are shown in bold and underlining,respectively. N_(T), total number of sequenced clones (includingsiblings) for this position.

FIG. 4 shows antigen-binding kinetics of Fab mutants at 37° C. Valuesfor k_(on) and k_(off) were measured by surface plasmon resonance (SPR)on a BIAcore 2000 or BIAcore 3000. These represent a mean of 4measurements at four different densities of HER2-ECD ranging from 86 to380 RU's. § indicates data for one mutant, Y92(V_(L))W, which showedpoor expression and HER2 binding, suggesting that this mutant wasmisfolded. Multiple mutants are M.3(N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W) and M.7(N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W+T94(V_(L))S+D98(V_(H))W+Y100a(V_(H))F+Y102(V_(H))V).

FIG. 5 shows the variability of selected residues in hu4D5-8. TheWu-Kabat variability parameter (V_(S)) for the phage selected results(solid) versus the natural variability of human Kappa light chains andhuman heavy chains (gray). Variability is calculated as follows:V_(S)=n_(as)/(N_(max)/N_(total)) where n_(an)=the number of differentamino acids (i.e. of the 20 possible) at a given position,N_(max)=occurrences of the most common amino acid at that position, andN_(total)=total number of amino acids at that position (Wu, T. T. &Kabat, E. A., J Exp Med 132(2), 211-50 (1970)).

FIG. 6 shows the binding affinities of Fab variants to HER2-ECD. Mutantsare compared to wild-type at each temperature indicated. Over thistemperature range WT becomes slightly weaker (ΔΔG=0.20) at highertemperatures. Differences in binding energies (ΔΔG) as compared withhu4D5-8 were calculated for each mutant using K_(D) values as shown inFIG. 4: (ΔΔG=ΔG(WT)−ΔG(mutant)=−RT ln (K_(D) ^((mutant))/K_(D)^((wild-type))). The order of mutants represented is the same for eachtemperature panel: (1) Y100a(V_(H))F; (2) T94(V_(L))S; (3) Y102(V_(H))V;(4) N30(V_(L))S; (5) H91(V_(L))F; (6)N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W; (7) the multiple mutantN30(V_(L))S+H91(V_(L))F+Y92(V_(L))W+T94(V_(L))S+D98(V_(H))W+Y100a(V_(H))F+Y102(V_(H))V;and (8) D98(V_(H))W.

FIG. 7 illustrates a comparison of sequence variability (A) and Ala-scanresults (B) on the hu4D5-8 structure. Residues selected from phagelibraries (A) fall into 3 categories: Class 1, low variability (residuesN30′, G99′, Y100a, W95, R50′, Y33, R58′, and Y56), Class 2, moderatevariability (residues Y92, H91′, T94′, F53′, Y49, Y55, and F100′), andClass 3, high variability (residues Y102 and D98′). (B) Alanine scanresults (Kelley, R. F. & O'Connell, M. P., Biochemistry 32(27):6828-35(1993)) showing residues with effects on K_(D) of 50-fold to 5000-fold(residues H91′, Y100a, W95, and R50′), 1.5 to 2 fold (residues Y92,N30′, F53′, Y49, F100′, and D98′), and <1.5-fold (including smallimprovements in K_(D)) (residues D28′, R66′, T31′, S50′, S52′, Y55,T93′, T94′, Y33, T32′, Y56, Y52, D31′, N54′, T53′, and K30′).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based on the identification of variants of ahumanized anti-HER2 antibody, hu4D5-8, having HER2 binding affinityequal to or greater than the parent antibody. These variants wereidentified from a set of Fab libraries in which nineteen positions inthe light and/or heavy variable domains were substituted with all 20amino acids. The positions were selected for substitutions based in parton alanine scanning mutagenesis of the hu4D5-8 variable regions.Sequence variability within the high-affinity HER2-binding site of thehu4D5-8 antibody was tested by constructing monovalently displayedFab-phage libraries, selecting for HER2 binding clones, and sequencing alarge sample (50-70 clones) from each library pool at a point in theselection process where a high level of overall diversity (minimalsiblings, that is occurrence of identical clones) was observed. Thebinding affinities of soluble Fab fragments were also tested. A singlemutant, D98(V_(H))W was found to have a 3-fold improvement in bindingaffinity over wild-type hu4D5-8 Fab.

Accordingly, the present invention concerns antibody variants,particularly anti-HER2 antibody variants.

1. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994). One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. Indeed, the presentinvention is in no way limited to the methods and materials described.For purposes of the present invention, the following terms are definedbelow.

Throughout the disclosure, the terms “ErbB2”, “ErbB2 receptor”,“c-Erb-B2”, and “HER2” are used interchangeably, and, unless otherwiseindicated, refer to a native sequence ErbB2 human polypeptide, or afunctional derivative thereof. “her2”, “erbB2” and “c-erb-B2” refer tothe corresponding human gene. The terms “native sequence” or “native” inthis context refer to a polypeptide having the sequence of a naturallyoccurring polypeptide, regardless its mode of preparation. Such nativesequence polypeptides can be isolated from nature or can be produced byrecombinant or synthetic means, or by any combination of these orsimilar methods.

Accordingly, “native” or “native sequence” HER2 polypeptides may beisolated from nature, produced by techniques of recombinant DNAtechnology, chemically synthesized, or produced by any combinations ofthese or similar methods. The amino acid sequence and encodingnucleotide sequence of a native human HER2 polypeptide is disclosed, forexample, in Semba et al., PNAS (USA) 82:6497-65)2 (1985) and Yamamoto etal., Nature 319:230-234 (1986) (GenBank accession number Xo3363). ErbB2comprises four domains (Domains 1-4). HER2 polypeptides from othernon-human animals, e.g. mammalian species are also well known in theart. “Functional derivatives” include amino acid sequence variants, andcovalent derivatives of the native polypeptides as long as they retain aqualitative biological activity of the corresponding native polypeptide.Amino acid sequence “variants” generally differ from a native sequencein the substitution, deletion and/or insertion of one or more aminoacids anywhere within a native amino acid sequence. Deletional variantsinclude fragments of the native polypeptides, and variants having N-and/or C-terminal truncations.

“Heregulin” (HRG) when used herein refers to a polypeptide whichactivates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e.induces phosphorylation of tyrosine residues in the complex upon bindingthereto). Various heregulin polypeptides encompassed by this term aredisclosed in Holmes et al., Science 256:1205-1210 (1992); WO 92/20798;Wen et al., Mol. Cell. Biol. 14(3):1909-1919 (1994) and Marchionni etal., Nature 362:312-318 (1993), for example. The term includesbiologically active fragments and/or variants of a naturally occurringHRG polypeptide, such as an EGF-like domain fragment thereof (e.g.HRGβ₁₇₇₋₂₄₄).

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term also includes, as equivalents, analogs of either DNA orRNA made from nucleotide analogs, and as applicable, single (sense orantisense) and double-stranded polynucleotides. An “isolated” nucleicacid molecule is a nucleic acid molecule that is identified andseparated from at least one contaminant nucleic acid molecule with whichit is ordinarily associated in the natural source of the nucleic acid.An isolated nucleic acid molecule is other than in the form or settingin which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the nucleic acid molecule as it existsin natural cells. However, an isolated nucleic acid molecule includes anucleic acid molecule contained in cells that ordinarily express theantibody where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. The term “expression vector” includes plasmids, cosmids orphages capable of synthesizing the subject HER2 protein encoded by therespective recombinant gene carried by the vector. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. In the present specification, “plasmid” and“vector” are used interchangeably, as the plasmid is the most commonlyused form of vector.

The term “transfection” refers to the introduction of a nucleic acid,e.g., an expression vector, into a recipient cell by nucleicacid-mediated gene transfer. “Transformation”, as used herein, refers toa process in which a cell's genotype is changed as a result of thecellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of HER2.

The term “non-human mammal” refers to all members of the class Mammaliaexcept humans. “Mammal” refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as mouse, rat, rabbit, pig, sheep, goat, cattle and higherprimates.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. The term “progeny” refers to any and all offspring of everygeneration subsequent to an originally transformed cell or cell line.Mutant progeny that have the same function or biological activity asscreened for in the originally transformed cell are included. Wheredistinct designations are intended, it will be clear from the context.

The term “antibody” herein is used in the broadest sense andspecifically covers intact antibodies, monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments, so long asthey exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

Antibodies specifically include “chimeric” antibodies in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-6855 (1984)). Chimeric antibodies of interest hereininclude primatized antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g. Old World Monkey, Apeetc) and human constant region sequences.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′)₂, Fabc, Fv), in which all or substantially all of thehypervariable loops correspond to those of a non-human immunoglobulinand all or substantially all of the FRs are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Ricchmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2,huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 andhuMAb4D5-8 (HERCEPTIN7) as described in Table 3 of U.S. Pat. No.5,821,337 expressly incorporated herein by reference; humanized 520C9(WO93/21319) and humanized 2C4 antibodies as described in copendingapplication Ser. No. 09/811,115, incorporated herein by reference.Throughout the disclosure, the terms “huMAb4D5-8” and “hu4D5-8” are usedinterchangeably.

The term “hypervariable region” when used herein refers to the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops. The hypervariable regioncomprises amino acid residues from a “complementarity determiningregion” or “CDR” (i.e. residues 24-34, 50-56, and 89-97 in the lightchain variable domain and 31-35, 50-65, and 95-102 in the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(i.e. residues 26-32, 50-52, and 91-96 in the light chain variabledomain and 26-32, 53-55, and 96-101 in the heavy chain variable domain;Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In both cases, thevariable domain residues are numbered according to Kabat et al., supra,as discussed in more detail below. “Framework” or “FR” residues arethose variable domain residues other than the residues in thehypervariable regions as herein defined.

A “parent antibody” or “wild-type” antibody is an antibody comprising anamino acid sequence which lacks one or more amino acid sequencealterations compared to an antibody variant as herein disclosed. Thus,the parent antibody generally has at least one hypervariable regionwhich differs in amino acid sequence from the amino acid sequence of thecorresponding hypervariable region of an antibody variant as hereindisclosed. The parent polypeptide may comprise a native sequence (i.e. anaturally occurring) antibody (including a naturally occurring allelicvariant), or an antibody with pre-existing amino acid sequencemodifications (such as insertions, deletions and/or other alterations)of a naturally occurring sequence. Preferably the parent antibody is achimeric, humanized or human antibody. For example, for purposes of theexamples disclosed below, the wild-type antibody hu4D5-8 is huMAb4D5-8,as described in U.S. Pat. No. 5,821,337, without any amino acidsubstitutions or other modifications. Throughout the disclosure, “wildtype,” “WT,” “wt,” and “parent” or “parental” antibody are usedinterchangeably.

As used herein, “antibody variant” or “variant antibody” refers to anantibody which has an amino acid sequence which differs from the aminoacid sequence of a parent antibody. Preferably, the antibody variantcomprises a heavy chain variable domain or a light chain variable domainhaving an amino acid sequence which is not found in nature. Suchvariants necessarily have less than 100% sequence identity or similaritywith the parent antibody. In a preferred embodiment, the antibodyvariant will have an amino acid sequence from about 75% to less than100% amino acid sequence identity or similarity with the amino acidsequence of either the heavy or light chain variable domain of theparent antibody, more preferably from about 80% to less than 100%, morepreferably from about 85% to less than 100%, more preferably from about90% to less than 100%, and most preferably from about 95% to less than100%. Identity or similarity with respect to this sequence is definedherein as the percentage of amino acid residues in the candidatesequence that are identical (i.e same residue) with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity. The antibodyvariant is generally one which comprises one or more amino acidalterations in or adjacent to one or more hypervariable regions thereof.

An “amino acid alteration” refers to a change in the amino acid sequenceof a predetermined amino acid sequence. Exemplary alterations includeinsertions, substitutions and deletions. An “amino acid substitution”refers to the replacement of an existing amino acid residue in apredetermined amino acid sequence; with another different amino acidresidue.

A “replacement” amino acid residue refers to an amino acid residue thatreplaces or substitutes another amino acid residue in an amino acidsequence. The replacement residue may be a naturally occurring ornon-naturally occurring amino acid residue.

An “amino acid insertion” refers to the introduction of one or moreamino acid residues into a predetermined amino acid sequence. The aminoacid insertion may comprise a “peptide insertion” in which case apeptide comprising two or more amino acid residues joined by peptidebond(s) is introduced into the predetermined amino acid sequence. Wherethe amino acid insertion involves insertion of a peptide, the insertedpeptide may be generated by random mutagenesis such that it has an aminoacid sequence which does not exist in nature. An amino acid alteration“adjacent a hypervariable region” refers to the introduction orsubstitution of one or more amino acid residues at the N-terminal and/orC-terminal end of a hypervariable region, such that at least one of theinserted or replacement amino acid residue(s) form a peptide bond withthe N-terminal or C-terminal amino acid residue of the hypervariableregion in question.

A “naturally occurring amino acid residue” is one encoded by the geneticcode, generally selected from the group consisting of: alanine (Ala);arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His);isoleucine (lie): leucine (Leu); lysine (Lys); methionine (Met);phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr);tryptophan (Trp); tyrosine (Tyr); and valine (Val).

A “non-naturally occurring amino acid residue” herein is an amino acidresidue other than those naturally occurring amino acid residues listedabove, which is able to covalently bind adjacent amino acid residues(s)in a polypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301-336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

Throughout this disclosure, reference is made to the numbering systemfrom Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987) and(1991). In these compendiums, Kabat lists many amino acid sequences forantibodies for each subclass, and lists the most commonly occurringamino acid for each residue position in that subclass. Kabat uses amethod for assigning a residue number to each amino acid in a listedsequence, and this method for assigning residue numbers has becomestandard in the field. The Kabat numbering scheme is followed in thisdescription. For purposes of this invention, to assign residue numbersto a candidate antibody amino acid sequence which is not included in theKabat compendium, one follows the following steps. Generally, thecandidate sequence is aligned with any immunoglobulin sequence or anyconsensus sequence in Kabat. Alignment may be done by hand, or bycomputer using commonly accepted computer programs; an example of such aprogram is the Align 2 program. Alignment may be facilitated by usingsome amino acid residues which are common to most Fab sequences. Forexample, the light and heavy chains each typically have two cysteineswhich have the same residue numbers; in V_(L) domain the two cysteinesare typically at residue numbers 23 and 88, and in the V_(H) domain thetwo cysteine residues are typically numbered 22 and 92. Frameworkresidues generally, but not always, have approximately the same numberof residues, however the CDRs will vary in size. For example, in thecase of a CDR from a candidate sequence which is longer than the CDR inthe sequence in Kabat to which it is aligned, typically suffixes areadded to the residue number to indicate the insertion of additionalresidues (see, e.g. residues 100abc in FIG. 1B). For candidate sequenceswhich, for example, align with a Kabat sequence for residues 34 and 36but have no residue between them to align with residue 35, the number 35is simply not assigned to a residue.

As described herein, particular amino acid residues may be substitutedwith other residues. The designation for a substitution variant hereinconsists of a letter followed by a number followed by a letter. Thefirst (leftmost) letter designates the amino acid in the wild-typeantibody. The number refers to the amino acid position where the aminoacid substitution is being made, and the second (right-hand) letterdesignates the amino acid that is used to replace the wild-type aminoacid at that position. In addition, a reference to the antibody lightchain variable domain (V_(L)) or heavy chain variable domain (V_(H)) maybe inserted following the number to indicate the specific location ofthe residue and/or substitution. For example, the hu4D5-8 variantslisted in FIG. 4 are designated with reference to the wild-type hu4D5-8antibody light chain and heavy chain variable region amino acidsequences (SEQ ID NOs: 1 and 2).

As used herein, an antibody with a “high-affinity” is an antibody havinga K_(D), or dissociation constant, in the nanomolar (nM) range orbetter. A K_(D) in the “nanomolar range or better” may be denoted by XnM, where X is a number less than about 10.

A molecule which “induces cell death” is one which causes a viable cellto become nonviable. The cell is generally one that expresses the ErbB2receptor, especially where the cell overexpresses the ErbB2 receptor.Preferably, the cell is a cancer cell, e.g. a breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic,prostate or bladder cancer cell. In vitro, the cell may be a SK-BR-3,BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell death in vitromay be determined in the absence of complement and immune effector cellsto distinguish cell death induced by antibody-dependent cell-mediatedcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus,the assay for cell death may be performed using heat inactivated serum(i.e. in the absence of complement) and in the absence of immuneeffector cells. To determine whether the molecule is able to induce celldeath, loss of membrane integrity as evaluated by uptake of propidiumiodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11(1995)) or 7AAD can be assessed relative to untreated cells. Preferredcell death-inducing antibodies are those which induce PI uptake in thePI uptake assay in BT474 cells (sec below).

A molecule which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one that overexpresses the ErbB2 receptor. Preferablythe cell is a tumor cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate orbladder cancer cell. In vitro, the cell may be a SK-BR-3, BT474, Calu 3cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Various methods areavailable for evaluating the cellular events associated with apoptosis.For example, phosphatidyl serine (PS) translocation can be measured byannexin binding; DNA fragmentation can be evaluated through DNAladdering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the molecule which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcells, in an annexin binding assay using BT474 cells. Sometimes thepro-apoptotic molecule will be one which further blocks ErbB ligandactivation of an ErbB receptor. In other situations, the molecule is onewhich does not significantly block ErbB ligand activation of an ErbBreceptor. Further, the molecule may induce apoptosis, without inducing alarge reduction in the percent of cells in S phase (e.g. one which onlyinduces about 0-10% reduction in the percent of these cells relative tocontrol).

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as the development or spread of cancer. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

The term “therapeutically effective amount” refers to an amount of amolecule effective to treat a disease or disorder in a mammal. In thecase of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the molecule may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. For cancertherapy, efficacy can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rate (RR).

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

An “ErbB-expressing” cancer or cancer comprising “ErbB-expressing cells”is a cancer comprising cells which have ErbB protein present at theircell surface. An “ErbB-expressing” cancer or cancer comprising“ErbB-expressing cells” is one which produces sufficient levels of ErbB2at the surface of cells thereof, such that an anti-ErbB2 antibody canbind thereto and have a therapeutic effect with respect to the cancer.

A cancer “characterized by excessive activation” of an ErbB receptor isone in which the extent of ErbB receptor activation in cancer cellssignificantly exceeds the level of activation of that receptor innon-cancerous cells of the same tissue type. Such excessive activationmay result from overexpression of the ErbB receptor and/or greater thannormal levels of an ErbB ligand available for activating the ErbBreceptor in the cancer cells. Such excessive activation may cause and/orbe caused by the malignant state of a cancer cell.

A cancer which “overexpresses” an ErbB receptor is one which hassignificantly higher levels of an ErbB receptor, such as HER2, at thecell surface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. ErbB receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the ErbB protein present on the surface of a cell(e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of ErbB-encoding nucleic acid inthe cell, e.g. via fluorescent in situ hybridization (FISH; seeWO98/45479 published October, 1998), Southern blotting, or polymerasechain reaction (PCR) techniques, such as real time quantitative PCR(RT-PCR). One may also study ErbB receptor overexpression by measuringshed antigen (e.g., ErbB extracellular domain) in a biological fluidsuch as serum (see, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990;WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar.28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Asidefrom the above assays, various in vive assays are available to theskilled practitioner. For example, one may expose cells within the bodyof the patient to an antibody which is optionally labeled with adetectable label, e.g. a radioactive isotope, and binding of theantibody to cells in the patient can be evaluated, e.g. by externalscanning for radioactivity or by analyzing a biopsy taken from a patientpreviously exposed to the antibody.

The tumors overexpressing HER2 may be rated by immunohistochemicalscores corresponding to the number of copies of HER2 molecules expressedper cell, and can been determined biochemically: 0=0-10,000 copies/cell,1+=at least about 200,000 copies/cell, 2+=at least about 500,000copies/cell, 3+=at least about 2,000,000 copies/cell. Overexpression ofHER2 at the 3+ level, which leads to ligand-independent activation ofthe tyrosine kinase (Hudziak et al., A., Mol Cell Biol 9(3):1165-72(1989)), occurs in approximately 30% of breast cancers, and in thesepatients, relapse-free survival and overall survival are diminished(Slamon et al., Science 244(4905):707-12 (1989); Slamon et al., Science235: 177-182 (1987)).

Conversely, a cancer which is “not characterized by overexpression ofthe ErbB2 receptor” is one which, in a diagnostic assay, does notexpress higher than normal levels of ErbB2 receptor compared to anoncancerous cell of the same tissue type.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2′=-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL⁷, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE7, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

2. Detailed Description

The present invention concerns antibody variants, preferably anti-HER2antibody variants. The variant antibodies may take a number of differentforms. For example, the antibodies may be, without limitation, intactantibodies, such as IgG1 antibodies, antibody fragments, such as a Fab,bispecific antibodies, humanized antibodies, or human antibodies.

The antibody variants preferably comprise one or more amino acidsubstitutions in the heavy chain variable domain and/or the light chainvariable domain. More preferably, the antibody variants comprise one ormore amino acid substitutions in the hypervariable regions of the heavychain variable domain and/or the light chain variable domain.

While the present invention contemplates single amino acid substitutionsaccording to the criteria herein, two or more substitutions may also becombined, e.g. from about two to about ten or about twenty substitutionsper variable domain (i.e. up to about twenty or about forty,respectively, amino acid substitutions for both variable domains). Thealterations described herein may be combined with other amino acidsequence alterations in the hypervariable regions or amino acid sequencealterations in other regions of the antibody.

Intact antibodies comprising the modified heavy and/or light chaindomains described herein may be made by methods well known in the art.For example, recombinant variant antibodies may be produced in hostcells such as E. coli cells, simian COS cells, Chinese Hamster Ovary(CHO) cells, or myeloma cells that do not otherwise produce antibodyprotein.

Alternatively, intact antibodies or antibody fragments can be isolatedfrom antibody phage libraries generated using the techniques described,for example, in McCafferty et al, Nature, 348:552-554 (1990).

a. Humanized Antibodies

Methods for producing humanized antibodies, particularly humanizedanti-HER2 antibodies are known. For example, production of a humanizedanti-HER2 antibody known as hu4D5-8, are described, in the examplesbelow and in U.S. Pat. No. 5,821,337, which is expressly incorporatedherein by reference.

This antibody was derived from a murine monoclonal antibody, 4D5 (Fendlyet au, Cancer Res 50(5):1550-8 (1990)), raised against the gene productof erbB2 known as p185 or HER2 (Slamon et al., Science 244(4905):707-12(1989)). The murine monoclonal antibody 4D5 and its uses are describedin PCT application WO 89106692 published 27 Jul. 1989. Murine antibody4D5 was deposited with the ATCC and designated ATCC CRL 10463.

Both hu4D5 and 4D5 demonstrate antiproliferative activity againstcarcinoma cells overexpressing p185^(HER2) (Carter et al., Proc NatlAcad Sci USA 89(10):4285-9 (1992b); Hudziak et al., Mol Cell Biol9(3):1165-72 (1989)). The IgG form of hu4D5-8 (Herceptin R; trastuzumab)is used as a therapeutic in the treatment of breast cancer (reviewed byMcKeage, K. & Perry, C. M., Drugs 62(1):209-43 (2002)).

In general, a humanized antibody preferably has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536(1988)), by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Example 1 below describes production of an exemplary humanizedanti-ErbB2 antibody. The humanized antibody herein may, for example,comprise nonhuman hypervariable region residues incorporated into ahuman variable heavy domain and may further comprise a framework region(FR) substitution at a position selected from the group consisting ofR66(V_(L)), A71(V_(H)), T73(V_(H)), A78(V_(H)), and S93(V_(H)) utilizingthe variable domain numbering system set forth in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). In oneembodiment, the humanized antibody comprises FR substitutions at two orall of positions R66(V_(L)), A71(V_(H)), T73(V_(H)), A78(V_(H)), andS93(V_(H)).

b. Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human anti-ErbB2 antibodies are described in U.S. Pat. No. 5,772,997issued Jun. 30, 1998 and WO 97/00271 published Jan. 3, 1997.

c. Antibody Fragments

Antibody fragments comprising the variant light and/or heavy chainvariable domains described herein are contemplated. Various techniqueshave been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992); and Brennan et al., Science,229:81 (1985)).

However, other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. For example, antibody fragmentscan now be produced directly by recombinant host cells. In oneembodiment, the antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). According to another approach, F(ab′)2fragments can be isolated directly from recombinant host cell culture.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)).

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.No. 5,587,458. The antibody fragment may also be a “linear antibody”,e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linearantibody fragments may be monospecific or bispecific.

d. Bispecific Antibodies

Bispecific antibodies that comprise the binding site of the anti-HER2antibody variants described herein are contemplated. Bispecificantibodies are antibodies that have binding specificities for at leasttwo different epitopes. Exemplary bispecific antibodies may bind to twodifferent epitopes of the ErbB2 protein. Other such antibodies maycombine an ErbB2 binding site with binding site(s) for EGFR, ErbB3and/or ErbB4. Alternatively, an anti-ErbB2 arm may be combined with anarm which binds to a triggering molecule on a leukocyte such as a T-cellreceptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR),such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focuscellular defense mechanisms to the ErbB2-expressing cell. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress ErbB2. WO 96/16673 describes a bispecificanti-ErbB2/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses abispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcαantibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches abispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gone fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

e. Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies are contemplated.For example, it may be desirable to improve the binding affinity and/orother biological properties of an antibody. Amino acid sequence variantsmay be prepared by introducing appropriate nucleotide changes into thenucleic acid encoding the antibody variant, or by peptide synthesis.Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibody variants. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processing ofthe antibody variants, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theantibodies that are preferred locations for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells,Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with a particular antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody variant with an N-terminal methionyl residue or the antibodyfused to a cytotoxic polypeptide. Other insertional variants of theantibody molecule include the fusion of the N- or C-terminus of theantibody to an enzyme (e.g. for ADEPT) or a polypeptide which increasesthe serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred conservative substitutions”.If such substitutions result in a change in biological activity, thenmore substantial changes, denominated “exemplary substitutions” in Table1, or as further described below in reference to amino acid classes, maybe introduced and the products screened. The most preferred amino acidsubstitution variants are described in the examples below.

TABLE 1 Original Preferred Conservative Residue Exemplary SubstitutionsSubstitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn(N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; alaser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H)asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leuLeu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in the biological properties of the antibodymay be accomplished by selecting substitutions that differ significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Naturally occurring residues may be divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody variants also may be substituted, generally with serine,to improve the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cystcine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) may be selected based on their biological properties. Assuch, variant(s) selected for further development may have improvedbiological properties relative to the parent antibody from which theyare generated, such as enhanced binding affinity. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display, discussed below.

It may also be desirable to modify the antibody of the invention withrespect to effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B., J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3.219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

f. Affinity Maturation

Affinity maturation can produce antibodies with improved affinity, incomparison to the parent antibody. Sequence diversity in naturallyoccurring antibodies arises in B-cells with the recombination ofselected diverse gene segments with imprecise cleavage events,nucleotide insertions, and secondary gene rearrangements, followedduring maturation of the immunoglobulin response by secondary generearrangements and point mutations. These changes serve to enhance thespecificity and effectiveness of the immune response through theselection of B-cell clones producing antibodies of increasing affinityand specificity.

The affinity maturation process has been effectively mimicked in vitrousing antibody diversity libraries displayed on phage, yeast, or otherhosts (reviewed by Hoogenboom, H. R. & Chames, P., Immunol Today21(8):371-8 (2000); Maynard, J. & Georgiou, G., Annu Rev Biomed Eng2:339-76 (2000); Rader, C. & Barbas, C. F., 3rd., Curr Opin Biotechnol8(4):503-8 (1997)). In particular, phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993).

In one approach, nucleotide sequences coding for antibody hypervariableregion sites of interest are mutated to generate all possible aminosubstitutions at each site. The mutated sequences are cloned in-frameinto either a major or minor coat protein gene of a filamentousbacteriophage and displayed as functional antibody fragments on thesurface of the phage particle.

The display is monovalent if a single antibody fragment is displayed perphage cell. Monovalent display can be accomplished with the use ofphagemid and helper phage as described, for example, in Lowman, H. B.,Methods Mol Biol 87:249-64 (1998). A preferred phage is M13 and displayis preferably as a fusion protein with coat protein 3 as described inLowman et. al., supra. Other suitable phage include fl and fdfilamentous phage. Fusion protein display with other virus coat proteinsis also known and may be used in this invention. See U.S. Pat. No.5,223,409.

Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell. In particular, the phage-displayed variantsmay be screened for their biological activity (e.g. binding affinity) asherein disclosed. Subsequent selection of variants with particularbiological properties (e.g. high binding affinity) and continuedrescreening of and reselection from the population of selected variantsallows identification of variants with improvements in the biologicalactivity screened for, such as increased affinity for a particularantigen.

Alanine scanning mutagenesis can be performed to identify candidatehypervariable region sites for modification. Those hypervariable regionresidues identified as contributing significantly to antigen binding arecandidates for modification. Alternatively, or additionally, it may bebeneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and an antigen.Such contact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development, asdiscussed above.

The process of affinity maturation can produce striking improvements inaffinity compared to the parental antibody. A study in the mouse showed10-10³-fold improvements in K_(D) during in vivo affinity maturation(Foote, J. & Milstein, C., Nature 352(6335):530-2 (1991)). Using ayeast-displayed scFv library, Wittrup and coworkers were able to improvethe binding affinity of an antibody >1000-fold, to 48 fM (K_(D)=4.8×10¹⁴M; (Boder et al., Proc Natl Acad Sci USA 97(20):10701-5 (2000)). Equallystriking is the fact that a small number of mutations can sometimesaffect these changes. For example, antigen-binding affinities wereimproved by 16-fold in a CDR-L3 point mutant of another anti-erbB2antibody (Schier et al., J Mol Biol 263(4):551-67 (1996)), 14-fold in aCDR-H3 point mutant of an anti-VEGF (Chen et al., J Mol Biol293(4):865-81 (1999)), and 8-fold in a CDR-H3 point mutant of ananti-gp120 antibody (Barbas et al., Proc Natl Acad Sci USA 91(9):3809-13(1994)).

g. Screening for Antibodies with Desired Properties

After generating variant antibodies, one may further select those withparticular biological characteristics, as desired.

For example, one may screen for antibodies with a desired bindingaffinity. As discussed below in the example, phage displayed Fablibraries may be sorted based on binding affinity. Briefly, antibodyfragments derived from particular antibodies may be phage displayed andorganized into libraries. The libraries may then be subjected toincreasingly stringent rounds of antigen-binding selection usingdecreasing concentrations of antigen.

In addition, one may identify high affinity antibodies by determiningthe binding affinity and kinetics of a population of antibodies. In oneembodiment, surface plasmon resonance (SPR) binding affinitymeasurements may be taken, as described in the examples below. Briefly,antibody fragments are derived from the antibodies of interest. ABIAcore-2000 or BIAcore-3000 real-time kinetic interaction analysissystem (Biacore Inc., Piscataway, N.J.) may then be used to determineassociation (k_(on)) and dissociation (k_(off)) constants (Karlsson, R.,Michaelsson, A. & Mattsson, L., J Immunol Methods 145(1-2):229-40(1991)) of the antibody fragments in binding interactions withimmobilized antigen, according the manufacture's instructions. Anequilibrium constant, K_(D), may be calculated from k_(off)/k_(on), asknown in the art. Free energy differences, as compared with wild-typeantibody may be calculated as described (Wells, J. A., Biochemistry29(37), 8509-17 (1990)): ΔΔG=−RT ln (K_(D) ^((mutant))/K_(D)^((wild-type)).

Furthermore, in one embodiment, to identify growth inhibitory anti-ErbB2antibodies, one may screen for antibodies which inhibit the growth ofcancer cells which overexpress ErbB2. In one embodiment, a growthinhibitory antibody is able to inhibit growth of SK-BR-3 cells in cellculture by about 20-100% and preferably by about 50-100%/o at anantibody concentration of about 0.5 to 30 μg/ml. To identify suchantibodies, the SK-BR-3 assay described in U.S. Pat. No. 5,677,171 canbe performed. According to this assay, SK-BR-3 cells are grown in a 1:1mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum,glutamine and penicillin streptomycin. The SK-BR-3 cells are plated at20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish). 0.5 to 30μg/ml of the anti-ErbB2 antibody is added per dish. After six days, thenumber of cells, compared to untreated cells are counted using anelectronic COULTER™ cell counter. Those antibodies which inhibit growthof the SK-BR-3 cells by about 20-100%, and more preferably about 50-100%may be selected as growth inhibitory antibodies.

To select for variant antibodies which induce cell death, loss ofmembrane integrity as indicated by, e.g., PI, trypan blue or 7AAD uptakemay be assessed relative to control. The preferred assay is the PIuptake assay using BT474 cells. According to this assay, BT474 cells(which can be obtained from the American Type Culture Collection(Rockville, Md.)) are cultured in Dulbecco's Modified Eagle Medium(D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS(Hyclone) and 2 mM L-glutamine. (Thus, the assay is performed in theabsence of complement and immune effector cells). The BT474 cells areseeded at a density of 3×10⁶ per dish in 100×20 mm dishes and allowed toattach overnight. The medium is then removed and replaced with freshmedium alone or medium containing 10 μg/ml of the appropriate monoclonalantibody. The cells are incubated for a 3 day time period. Followingeach treatment, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged at 1200 rpm for 5 minutes at4° C., the pellet resuspended in 3 ml ice cold Ca²⁺ binding buffer (10mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group)for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samplesmay be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Those antibodies which inducestatistically significant levels of cell death as determined by PIuptake may be selected as cell death-inducing antibodies.

Antibodies which induce apoptosis may also be selected. An annexinbinding assay using BT474 cells may be used to identify theseantibodies. The BT474 cells are cultured and seeded in dishes asdiscussed in the preceding paragraph. The medium is then removed andreplaced with fresh medium alone or medium containing 10 μg/ml of themonoclonal antibody. Following a three day incubation period, monolayersare washed with PBS and detached by trypsinization. Cells are thencentrifuged, resuspended in Ca²⁺ binding buffer and aliquoted into tubesas discussed above for the cell death assay. Tubes then receive labeledannexin (e.g. annexin V-FTIC) (1 μg/ml). Samples may be analyzed using aFACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BectonDickinson). Those antibodies which induce statistically significantlevels of annexin binding relative to control are selected asapoptosis-inducing antibodies.

In addition to the annexin binding assay, a DNA staining assay usingBT474 cells may be used to indentify antibodies that induce apoptosis.In order to perform this assay, BT474 cells which have been treated withthe antibody of interest, as described in the preceding two paragraphs,are incubated with 9 μg/ml HOECHST 33342™ for 2 hr at 37° C., thenanalyzed on an EPICS ELITE™ flow cytometer (Coulter Corporation) usingMODFIT LT™ software (Verity Software House). Antibodies which induce achange in the percentage of apoptotic cells which is 2 fold or greater(and preferably 3 fold or greater) than untreated cells (up to 100%apoptotic cells) may be selected as pro-apoptotic antibodies using thisassay.

In another embodiment, an antibody which blocks ligand activation of anErbB receptor may be selected by determining the ability of the antibodyto block ErbB ligand binding to cells expressing the ErbB receptor (e.g.in conjugation with another ErbB receptor with which the ErbB receptorof interest forms an ErbB hetero-oligomer). For example, cells naturallyexpressing, or transfected to express, ErbB receptors of the ErbBhetero-oligomer may be incubated with the antibody and then exposed tolabeled ErbB ligand. The ability of the anti-ErbB2 antibody to blockligand binding to the ErbB receptor in the ErbB hetero-oligomer may thenbe evaluated.

For example, inhibition of HRG binding to MCF7 breast tumor cell linesby anti-ErbB2 antibodies may be performed using monolayer MCF7 cultureson ice in a 24-well-plate format essentially as described in Example 1below. Anti-ErbB2 monoclonal antibodies may be added to each well andincubated for 30 minutes. ¹²⁵I-labeled rHRGβ1₁₇₇₋₂₂₄ (25 pm) may then beadded, and the incubation may be continued for 4 to 16 hours. Doseresponse curves may be prepared and an IC₅₀ value may be calculated forthe antibody of interest. In one embodiment, the antibody which blocksligand activation of an ErbB receptor will have an IC₅₀ for inhibitingHRG binding to MCF7 cells in this assay of about 50 nM or less, morepreferably 10 nM or less. Where the antibody is an antibody fragmentsuch as a Fab fragment, the IC₅₀ for inhibiting HRG binding to MCF7cells in this assay may, for example, be about 100 nM or less, morepreferably 50 nM or less.

Alternatively, or additionally, the ability of anti-ErbB2 antibodyvariants to block ErbB ligand-stimulated tyrosine phosphorylation of anErbB receptor present in an ErbB hetero-oligomer may be assessed. Forexample, cells endogenously expressing the ErbB receptors or transfectedto expressed them may be incubated with the antibody and then assayedfor ErbB ligand-dependent tyrosine phosphorylation activity using anantiphosphotyrosine monoclonal antibody (which is optionally conjugatedwith a detectable label). The kinase receptor activation assay describedin U.S. Pat. No. 5,766,863 is also available for determining ErbBreceptor activation and blocking of that activity by an antibody.

In one embodiment, one may screen for an antibody which inhibits HRGstimulation of p180 tyrosine phosphorylation in MCF7 cells. For example,the MCF7 cells may be plated in 24-well plates and monoclonal antibodiesto ErbB2 may be added to each well and incubated for 30 minutes at roomtemperature; then rHRGβ1₁₇₇₋₂₄₄ may be added to each well to a finalconcentration of 0.2 nM, and the incubation may be continued for 8minutes. Media may be aspirated from each well, and reactions may bestopped by the addition of 100 μl of SDS sample buffer (5% SDS, 25 mMDTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl) may beelectrophoresed on a 4-12% gradient gel (Novex) and thenelectrophoretically transferred to polyvinylidene difluoride membrane.Antiphosphotyrosine (at 1 μg/ml) immunoblots may be developed, and theintensity of the predominant reactive band at M_(r)˜180,000 may bequantified by reflectance densitometry. The antibody selected willpreferably significantly inhibit HRG stimulation of p180 tyrosinephosphorylation to about 0-35% of control in this assay. A dose-responsecurve for inhibition of HRG stimulation of p180 tyrosine phosphorylationas determined by reflectance densitometry may be prepared and an IC₅₀for the antibody of interest may be calculated. In one embodiment, theantibody which blocks ligand activation of an ErbB receptor will have anIC₅₀ for inhibiting HRG stimulation of p180 tyrosine phosphorylation inthis assay of about 50 nM or less, more preferably 10 nM or less. Wherethe antibody is an antibody fragment such as a Fab fragment, the IC₅₀for inhibiting HRG stimulation of p180 tyrosine phosphorylation in thisassay may, for example, be about 100 nM or less, more preferably 50 nMor less.

One may also assess the growth inhibitory effects of an antibody onMDA-MB-175 cells, e.g, essentially as described in Schaefer et al.,Oncogene 15:1385-1394 (1997). According to this assay, MDA-MB-175 cellsmay be treated with the anti-ErbB2 monoclonal antibody (10 μg/mL) for 4days and stained with crystal violet. Incubation with an anti-ErbB2antibody may show a growth inhibitory effect on this cell line similarto that displayed by monoclonal antibody 2C4. In a further embodiment,exogenous HRG will not significantly reverse this inhibition.Preferably, the antibody will be able to inhibit cell proliferation ofMDA-MB-175 cells to a greater extent than monoclonal antibody 4D5, bothin the presence and absence of exogenous HRG.

In one embodiment, the anti-ErbB2 antibody variants of interest mayblock heregulin dependent association of ErbB2 with ErbB3 in both MCF7and SK-BR-3 cells as determined in a co-immunoprecipitation experimentsubstantially more effectively than monoclonal antibody 4D5.

To screen for antibodies which bind to an epitope on ErbB2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art.

The results obtained in the cell-based assays described above can thenbe followed by testing in animal, e.g. murine, models, and humanclinical trials. In particular, the ability of an antibody variant totreat ErbB2 overexpressing tumors can be demonstrated in the transgenicmouse model disclosed in co-pending application Ser. No. 09/811,115.

h. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins,maytansinoids, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide). In a preferredembodiment, the antibody is conjugated to a maytansinoid as described incopending application Ser. No. 09/811,123.

i. Pharmaceutical Formulations

Therapeutic formulations of the antibody variants used in accordancewith the present invention are prepared by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-ErbB2 antibodyformulations are described in WO 97/04801, expressly incorporated hereinby reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies orantibody conjugates which bind to EGFR, ErbB2 (e.g. an antibody whichbinds a different epitope on ErbB2), ErbB3, ErbB4, or vascularendothelial factor (VEGF) in the one formulation. Alternatively, oradditionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

In one embodiment, the formulation comprises 5 mg/ml variant hu4D5-8,100 mg/ml sucrose, 0.1% polysorbate 20 and 10 mM sodium succinate at pH5.0.

Treatment with anti-ErbB2 antibody variants.

It is contemplated that, according to the present invention, anti-ErbB2antibody variants may be used to treat various diseases or disorders.Exemplary conditions or disorders include benign or malignant tumors;leukemias and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologicdisorders.

Generally, the disease or disorder to be treated is cancer. Examples ofcancer to be treated herein include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

Preferably, antibody variants are used to treat breast cancer. Thecancer will comprise ErbB-expressing cells, such that an anti-ErbBantibody herein is able to bind to the cancer, and will be typicallycharacterized by overexpression of the ErbB receptor. In a preferredembodiment, the cancer comprises ErbB2-expressing cells, even morepreferably, cells which are characterized by overexpression of the ErbB2receptor. To determine ErbB, e.g. ErbB2 expression in the cancer,various diagnostic/prognostic assays are available. In one embodiment,ErbB2 overexpression may be analyzed by immuno-histochemistry (IHC),e.g. using the HERCEPTEST® (Dako). Parrafin embedded tissue sectionsfrom a tumor biopsy may be subjected to the IHC assay and accorded aErbB2 protein staining intensity criteria as follows:

-   -   Score 0 no staining is observed or membrane staining is observed        in less than 10% of tumor cells.    -   Score 1+a faint/barely perceptible membrane staining is detected        in more than 10% of the tumor cells. The cells are only stained        in part of their membrane.    -   Score 2+a weak to moderate complete membrane staining is        observed in more than 10% of the tumor cells.    -   Score 3+a moderate to strong complete membrane staining is        observed in more than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for ErbB2 overexpression assessment maybe characterized as not overexpressing ErbB2, whereas those tumors with2+ or 3+ scores may be characterized as overexpressing ErbB2.

Alternatively, or additionally, fluorescence in situ hybridization(FISH) assays such as the INFORM™ (sold by Ventana, Arizona) orPATHVISION™ (Vysis, Illinois) assays may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of ErbB2 overexpression in the tumor. In comparison with theTHC assay, the FISH assay, which measures her2 gene amplification, seemsto correlate better with response of patients to treatment withanti-HER2 antibodies, and is currently considered to be the preferredassay to identify patients likely to benefit from anti-HER2 antibodytreatment (e.g. treatment with commercially available HERCEPTIN™) ortreatment with the variants of the present invention.

Preferably, the variants of the present invention and/or the ErbB, e.g.ErbB2, protein to which they are bound are internalized by the cell,resulting in increased therapeutic efficacy of the variant in killingthe cancer cell to which they bind. In a preferred embodiment, acytotoxic agent, such as a maytansinoid, targets or interferes withnucleic acid in the cancer cell.

The anti-ErbB antibody variants are administered to a mammal, preferablyto a human patient in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Other therapeutic regimens may be combined with the administration ofthe anti-ErbB antibody variants. The combined administration includescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities.

In one embodiment, the patient is treated with two or more differentanti-ErbB antibodies, at least one of which is in the form of a variant.For example, the patient may be treated with a first anti-ErbB2 antibodyvariant in which the antibody is growth inhibitory, and a secondanti-ErbB2 antibody or antibody-immunoconjugate, e.g. anantibody-maytansinoid conjugate, which blocks ligand activation of anErbB receptor (e.g. 2C4 or a humanized and/or affinity matured variantthereof) or induces apoptosis of an ErbB2-overexpressing cell (e.g. 7C2,7F3 or humanized and/or affinity matured variants thereof). In anotherembodiment, the treatment involves the administration of antibodies thatspecifically bind two or more different ErbB receptors, such as, forexample, ErbB2 and EGFR receptors, where at least one of the anti-ErbBantibodies is a hu4D5-8 variant. Preferably such combined therapyresults in a synergistic therapeutic effect.

It may also be desirable to combine administration of the anti-ErbBantibody variants, with administration of an antibody directed againstanother tumor-associated antigen, which is not member of the ErbB familyof receptors. The other antibody in this case may, for example, bind tovascular endothelial growth factor (VEGF), and may be in the form of amaytansinoid conjugate, or another immunoconjugate.

In one embodiment, the treatment of the present invention involves thecombined administration of an anti-ErbB2 antibody variant (or variants)and one or more chemotherapeutic agents or growth inhibitory agents,including coadministration of cocktails of different chemotherapeuticagents. Preferred chemotherapeutic agents include taxanes (such aspaclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

In a preferred embodiment, the treatment is initiated with an anti-ErbBantibody variant, followed by maintenance treatment with an parentalanti-ErbB antibody.

The antibody variants may be combined with an anti-hormonal compound;e.g., an anti-estrogen compound such as tamoxifen; an anti-progesteronesuch as onapristone (see, EP 616 812); or an anti-androgen such asflutamide, in dosages known for such molecules. Where the cancer to betreated is hormone independent cancer, the patient may previously havebeen subjected to anti-hormonal therapy and, after the cancer becomeshormone independent, the anti-ErbB2 antibody (and optionally otheragents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also coadminister a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokincs to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the agent and anti-ErbB2 antibody.

For the prevention or treatment of disease, the appropriate dosage of anantibody variant will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibodyvariant is suitably administered to the patient at one time or over aseries of treatments. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. The progress of thistherapy is easily monitored by conventional techniques and assays.

j. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an antibody variant, according to thepresent teachings. In one embodiment, the container is a 10 cc vialcontaining 10 mL of a solution comprising an antibody variant describedherein.

The label or package insert indicates that the composition is used fortreating the condition of choice, such as cancer. In a preferredembodiment the label or package inserts indicate that the composition isused for treating breast cancer. In another embodiment, the label orpackage inserts indicates that the composition comprising a variantantibody which binds ErbB2 can be used to treat cancer which expressesan ErbB receptor selected from the group consisting of epidermal growthfactor receptor (EGFR), ErbB2, ErbB3 and ErbB4, preferably EGFR. Inaddition, the label or package insert may indicate that the patient tobe treated is one having cancer characterized by excessive activation ofan ErbB receptor selected from EGFR, ErbB2, ErbB3 or ErbB4. For example,the cancer may be one which overexpresses one of these receptors and/orwhich overexpresses an ErbB ligand (such as TGF-α). The label or packageinsert may also indicate that the composition can be used to treatcancer, wherein the cancer is not characterized by overexpression of theErbB2 receptor. In other embodiments, the package insert may indicatethat the composition can also be used to treat hormone independentcancer, prostate cancer, colon cancer or colorectal cancer.

Moreover, the article of manufacture may comprise (a) a first containerwith a composition contained therein, wherein the composition comprisesan antibody variant which binds ErbB2 and inhibits growth of cancercells which overexpress ErbB2; and (b) a second container with acomposition contained therein, wherein the composition comprises asecond antibody which binds ErbB2 and blocks ligand activation of anErbB receptor, or a conjugate of this second antibody with amaytansinoid. The article of manufacture in this embodiment of theinvention may further comprise a package insert indicating that thefirst and second compositions can be used to treat cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Further details of the invention are illustrated in the followingnon-limiting examples.

Example 1 Production, Characterization and Humanization of Anti-ErbB2Monoclonal Antibody 4D5

The murine monoclonal antibody 4D5 which specifically binds theextracellular domain of ErbB2 was produced as described in Fendly etal., Cancer Res 50(5):1550-8 (1990). Briefly, NIH 3T3/HER2-3₄₀₀ cells(expressing approximately 1×10⁵ ErbB2 molecules/cell) produced asdescribed in Hudziak et al., Mol Cell Biol 9(3):1165-72 (1989) wereharvested with phosphate buffered saline (PBS) containing 25 mM EDTA andused to immunize BALB/c mice. The mice were given injections i.p. of 10⁷cells in 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with antisera thatimmunoprecipitated ³²P-labeled ErbB2 were given i.p. injections of awheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract onweeks 9 and 13. This was followed by an i.v. injection of 0.1 ml of theErbB2 preparation and the splenocytes were fused with mouse myeloma lineX63-Ag8.653. Hybridoma supernatants were screened for ErbB2-binding byELISA and radioimmunoprecipitation.

Epitope Mapping and Characterization

The ErbB2 epitope bound by monoclonal antibody 4D5 was determined bycompetitive binding analysis (Fendly et al., Cancer Res 50(5):1550-8(1990)). Cross-blocking studies were done by direct fluorescence onintact cells using the PANDEX™ Screen Machine to quantitatefluorescence. The monoclonal antibody was conjugated with fluoresceinisothiocyanate (FITC), using established procedures (Wofsy et al.Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi(eds.) San Francisco: W. J. Freeman Co. (1980)). Confluent monolayers ofNIH 3T3/HER2-3₄₀₀ cells were trypsinized, washed once, and resuspendedat 1.75×10⁶ cell/ml in cold PBS containing 0.5% bovine serum albumin(BSA) and 0.1% NaN₃. A final concentration of 1% latex particles (IDC,Portland, Oreg.) was added to reduce clogging of the PANDEX™ platemembranes. Cells in suspension, 20 μl, and 20 μl of purified monoclonalantibodies (100 μg/ml to 0.1 μg/ml) were added to the PANDEX™ platewells and incubated on ice for 30 minutes. A predetermined dilution ofthe FITC-labeled monoclonal antibody in 20 μl was added to each well,incubated for 30 minutes, washed, and the fluorescence was quantitatedby the PANDEX™. Monoclonal antibodies were considered to share anepitope if each blocked binding of the other by 50% or greater incomparison to an irrelevant monoclonal antibody control. In thisexperiment, monoclonal antibody 4D5 was assigned epitope I (amino acidresidues from about 529 to about 625, inclusive, within the ErbB2extracellular domain (residues 22 to about 645, inclusive).

The murine monoclonal anti-HER2 antibody 4D5 inhibits the growth ofbreast cancer cell lines. The growth inhibitory characteristics ofmonoclonal antibody 4D5 were evaluated using the breast tumor cell line,SK-BR-3 (see Hudziak et al., Mol Cell Biol 9(3):1165-72 (1989)).Briefly, SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsinand suspended in complete medium at a density of 4×10⁵ cells per ml.Aliquots of 100 μl (4×10⁴ cells) were plated into 96-well microdilutionplates, the cells were allowed to adhere, and 100 μl of media alone ormedia containing monoclonal antibody (final concentration 5 μg/ml) wasthen added. After 72 hours, plates were washed twice with PBS (pH 7.5),stained with crystal violet (0.5% in methanol), and analyzed forrelative cell proliferation as described in Sugarman et al. Science230:943-945 (1985). Monoclonal antibody 4D5 inhibited SK-BR-3 relativecell proliferation by about 56%.

Monoclonal antibody 4D5 was also evaluated for its ability to inhibitHRG-stimulated tyrosine phosphorylation of proteins in the M_(r) 180,000range from whole-cell lysates of MCF7 cells (Lewis et al. CancerResearch 56:1457-1465 (1996)). MCF7 cells are reported to express allknown ErbB receptors, but at relatively low levels. Since ErbB2, ErbB3,and ErbB4 have nearly identical molecular sizes, it is not possible todiscern which protein is becoming tyrosine phosphorylated whenwhole-cell lysates are evaluated by Western blot analysis. However,these cells are ideal for HRG tyrosine phosphorylation assays becauseunder the assay conditions used, in the absence of exogenously addedHRG, they exhibit low to undetectable levels of tyrosine phosphorylationproteins in the M_(r) 180,000 range.

MCF7 cells were plated in 24-well plates and monoclonal antibodies toErbB2 were added to each well and incubated for 30 minutes at roomtemperature; then rHRGβ1₁₇₇₋₂₄₄ was added to each well to a finalconcentration of 0.2 nM, and the incubation was continued for 8 minutes.Media was carefully aspirated from each well, and reactions were stoppedby the addition of 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and25 mM Tris-HCl, pH 6.8). Each sample (25 μl) was electrophoresed on a4-12% gradient gel (Novex) and then electrophoretically transferred topolyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from UBI,used at 1 μg/ml) immunoblots were developed, and the intensity of thepredominant reactive band at M_(r)−180,000 was quantified by reflectancedensitometry, as described previously (Holmes et al. Science256:1205-1210 (1992); Sliwkowski et al. J. Biol. Chem. 269:14661-14665(1994))

Monoclonal antibody 4D5 significantly inhibited the generation ofaHRG-induced tyrosine phosphorylation signal at M_(r) 180,000. In theabsence of HRG, but was unable to stimulate tyrosine phosphorylation ofproteins in the M_(r) 180,000 range. Also, this antibody does notcross-react with EGFR (Fendly et al., Cancer Res 50(5):1550-8 (1990)),ErbB3, or ErbB4. Monoclonal antibody 4D5 was able to block HRGstimulation of tyrosine phosphorylation by −50%.

The growth inhibitory effect of monoclonal antibody 4D5 on MDA-MB-175and SK-BR-3 cells in the presence or absence of exogenous rHRGβ1 wasassessed (Schaefer et al., Oncogene 15:1385-1394 (1997)). ErbB2 levelsin MDA-MB-175 cells are 4-6 times higher than the level found in normalbreast epithelial cells and the ErbB2-ErbB4 receptor is constitutivelytyrosine phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 wasable to inhibit cell proliferation of MDA-MB-175 cells, both in thepresence and absence of exogenous HRG. Inhibition of cell proliferationby 4D5 is dependent on the ErbB2 expression level (Lewis et al., CancerImmunol. Immunother. 37:255-263 (1993)). A maximum inhibition of 66% inSK-BR-3 cells could be detected. However this effect could be overcomeby exogenous HRG.

Humanization

The murine monoclonal antibody 4D5 was humanized, using a novel “geneconversion mutagenesis” strategy, as described in U.S. Pat. No.5,821,337, the entire disclosure of which is hereby expresslyincorporated by reference. The humanized monoclonal antibody 4D5 used inthe following experiments is the antibody variant designated as hu4D5-8in that patent. Hu4D5-8 comprises a light chain variable domain (V_(L))(SEQ ID NO: 1), and a heavy chain variable domain (V_(H)) (SEQ ID NO:2). Within the light chain variable domain of SEQ ID NO:1 are threehypervariable regions; V_(L)-hypervariable region 1, comprising aminoacids RASQDVNTAVA (SEQ ID NO: 19); V_(L)-hypervariable region 2comprising amino acids SASFLYS (SEQ ID NO: 20); and V_(L)-hypervariableregion 3 comprising amino acids QQHYTTPPT (SEQ ID NO: 21). Similarly,there are three hypervariable regions within the heavy chain variabledomain of SEQ ID NO: 2: V_(H)-hypervariable region 1 comprising aminoacids GFNIKDTYIH (SEQ ID NO: 22), V_(H)-hypervariable region 2comprising amino acids RIYPTNGYTRYADSVKG (SEQ ID NO: 23); andV_(H)-hypervariable region 3 comprising amino acids WGGDGFYAMDY (SEQ IDNO: 24).

Example 2 HU4D5-8 Variants

Recognition of an antibody often involves a subset of the hypervariableregion residues with contacts at the center of the antigen-combiningsite (see Schier et al., J Mol Biol 263(4):551-67 (1996)). This is thecase in the recognition of the tumor antigen HER2 by the humanizedantibody known as hu4D5. Phage display allowed exploration of theoverall variability of the binding site, revealing positions at whichfurther substitutions might be made to improve affinity. The amino acidsequences of the light and heavy chains of hu4D5-8, along with CDRresidues, are shown in FIGS. 1A and 1B, respectively.

Sequence variability within the high-affinity HER2-binding site of thehu4D5-8 antibody was tested by constructing monovalently displayedFab-phage libraries, selecting for HER2 binding clones, and sequencing alarge sample (50-70 clones) from each library pool at a point in theselection process where a high level of overall diversity (minimalsiblings, that is occurrence of identical clones) was observed.

Selection of CDR Residues for Substitutions

Design of the phage libraries centered on four key residues from analanine scan study (Kelley, R. F. & O'Connell, M. P., Biochemistry32(27):6828-35 (1993)). These included CDR residues both in thelight-chain (V_(L)) and in the heavy-chain (V_(H)) variable domains:H91(V_(L)), R50(V_(H)), W95(V_(H)), and Y100a(V_(H)) (Kelley, R. F. &O'Connell, M. P., Biochemistry 32(27):6828-35 (1993)). Also selected forsubstitutions were additional surface-exposed residues that wereproximal to the V_(L):V_(H) interface, near the center of the antigencombining site, based on inspection of the hu4D5-8 crystal structure(Eigenbrot et al., J Mol Biol 229(4):969-95 (1993)). Residues known tobe important for the main chain conformation, or canonical structure,(Chothia et al., Nature 342(6252):877-83 (1989)) were omitted from thelibraries. In order to achieve adequate representation of all variants,the targeted positions were divided into five libraries, each consistingof a small cluster of surface positions, with no more than sevenresidues targeted in each. Each library, except for one targeting fiveresidues in CDR-H3, allowed variation of residues from both V_(L) andV_(H)(FIG. 2). Some residues were represented in more than one libraryin order to test for context effects and allow for covariation withother proximal positions.

Oligonucleotides for Use In Site-Directed Mutagenesis

A total of 19 residues of hu4D5-8 were randomized using site-directedmutagenesis with degenerate NNS codons (N=A, G, T or C; S=G or C) thatencode all 20 amino acids. Site-directed mutagenesis was carried outusing the following deoxyoligonucleotides: Lib1.1 GCC AGT CAG GAT GTGNNS ACT GCT GTA GCC TGG (SEQ ID NO: 3); Lib1.2 CT TAT TAC TGT CAG CAANNS NNS ACT ACT CCT CCC ACG (SEQ ID NO: 4); Lib1.3 C CTG GAA TGG GTI GCANNS ATT TAT CCT ACG AAT GG (SEQ ID NO: 5); Lib1.4 C TAT TAT TGT TCT AGANNS GGA GGG GAC NNS TTC NNS GCT ATG GAC TAC TGG GG (SEQ ID NO: 6);Lib2.1 CCG AAA CTA CTG ATT NNS TCG GCA TCC NNS CTC TAC TCT GGA GTC (SEQID NO: 7); Lib2.2 C GCA ACT TAT TAC TGT CAG CAA NNS TAT ACT ACT CCT CCC(SEQ ID NO: 8); Lib2.3 GT TCT AGA TGG GGA GGG NNS NNS NNS NNS GCT ATGGAC TAC TGG G (SEQ ID NO: 9); Lib3.1 C AAC ATT AAA GAC ACC NNS ATA CACTOG GTG CGT C (SEQ ID NO: 10); Lib3.2 G GGC CTG GAA TGOG GTT GCA NNS ATTTAT CCT ACG AAT GGT NNS ACT NNS TAT GCC GAT AGC G (SEQ ID NO: 11);Lib3.3 C TAT TAT TOT TCT AGA NNS GGA GGG GAC GGC TTC (SEQ ID NO: 12);Lib3.4 CAG CAA CAT TAT ACT NNS CCT CCC ACG TTC GGA CA (SEQ ID NO: 13);Lib4.1 G CGT GCT GAG GAC ACT GCC GTC TAT TAT TGT TCT AGA TGG NNS NNS NNSNNS NNS TAT GCT ATG GAC TAC TGG GGT CAA GG (SEQ ID NO: 14); Lib5.1 CCGAAA CTA CTG ATT NNS TCG GCA TCC NNS CTC NNS TCT GGA GTC CCT TCT CGC (SEQID NO: 15); Lib5.2 GG GGA GGG GAC GGC NNS TAT GCT ATG GAC NNS TGG GGTCAA GGA ACC (SEQ ID NO: 16).

Oligonucleotides used to sequence the selected phage were TGT AAA ACGACG GCC AGT CCG TTT AGG TGT TIT CAC GAG CAC T (SEQ ID NO: 17) and CAGGAA ACA GCT ATG ACC GTT CCA CGA CAC CGT CAC CGG TTC (SEQ ID NO: 18).

Construction of hu4D5-8 Phage Libraries

The hu4D5-8 phagemid (564/11) was made by fusing the light and heavychains of the Fab (Kelley et al., Biochemistry 31(24):5434-41 (1992)) toa truncated form of g3, encoding one of the M13 phage coat proteins.

The hu4D5 libraries were constructed as described in previous methods(Sidhu et al., Methods Enzymol 328: 333-63 (2000a); Lowman, H. B.,Methods Mol Biol 87:249-64 (1998)). For each library, the template was amodified version of phagemid 564/11 that contained stop codons (TAA)introduced at positions where amino acids were to be mutated. Adifferent stop template was made for each library. The stop templatesand mutagenic oligos described in the previous section were used instandard Kunkel mutagenesis (Kunkel et al., Methods Enzymol 204:125-39(1991)). Annealing of mutagenic oligos to the stop template repaired thestop codon and introduced the desired mutations. All libraries were onthe order of 10¹⁰, well beyond the theoretical diversities by 10 to1000-fold. This ensured that at least one copy of all mutations waspresent in each library.

Sorting of hu4D5-8 Phage Libraries

Phage were amplified in E. coli and subjected to increasingly stringentrounds of antigen-binding selection using decreasing concentrations ofHER2-ECD, starting at 10 nM and decreasing 10-fold in each round.

The phage libraries were selected by their ability to bind to the HER2receptor using a strategy similar to that previously described (Hawkinset al., J Mol Biol 226(3):889-96 (1992)). Library phage that bound tobiotinylated HER2-ECD antigen were captured with magnetic beads that hadbeen blocked with milk protein for 1 hour at 37° C. A preincubation ofphage with beads for 1 hour at 37° C. minimized nonspecific binding ofthe phage selected in each round. The beads bound to HER2-phagecomplexes were separated and washed five times during round 1, and 10times for all subsequent rounds of sorting. Phage were eluted from thebeads and neutralized with HCl. A portion of the eluted phage werepropagated in rapidly dividing XL-1 (Stratagene, La Jolla, Calif.) orSS320 (Sidhu et al., Methods Enzymol 328: 333-63 (2000b)) cells in thepresence of M13-VCS (Stratagene). Library size was determined by platingserial dilutions of cells onto agar. Library enrichment was determinedby comparing the number of phage isolated in the presence and in theabsence of antigen. Phage were otherwise treated identically with regardto pre-incubation, separation by magnetic beads, and wash steps.

In the initial round of selection, 1×10¹³ of library phage wereincubated with 10 nM antigen. The antigen concentration was thendecreased 10-fold during each subsequent round of screening. The phagesupernatants of individual clones were assayed for activity in a phageELISA (Lowman, H. B., Methods Mol Biol 87:249-64 (1998)) and showed thatmore than 50% were positive after the second round of selection. Alllibraries displayed enrichment by round 4, with selection using 0.01 nMantigen.

Sequencing and Analysis of Phage DNA

Phage were sequenced directly from cell-culture supernatants. A standardPCR reaction of the phage amplified the light and heavy chain of theFab. The forward and reverse M13 universal primer sequence was includedin the PCR primers so that the product could be easily sequenced withstandard primers. The sequences obtained were first analyzed in theprogram SGcount as previously described (Weiss et al., Proc Natl AcadSci USA 97(16), 8950-4 (2000)). Clones with sequence uncertainties wereremoved from the analysis. The remaining sequences were then filtered by(1) removing siblings, (2) normalizing for any codon bias that resultedfrom the use of an NNS codon, and (3) normalizing for the total numberof sequences, so that the results from different libraries could bedirectly compared. The number of clones analyzed for each library was asfollows: 71 from library-1, 82 from library-2, 71 from library-3, 74from library-4, and 57 from library-5.

Analysis of Variability within the Binding Site of hu4D5-8

In order to map sequence variability within the binding site of hu4D5-8systematically, the Wu-Kabat variability coefficient from the sequencedata was calculated. Variability (V_(S)) is the number of differentamino acids at a given position divided by the frequency of the mostcommon amino acid at that position (Wu, T. T. & Kabat, E. A., J Exp Med132(2), 211-50 (1970)).

Clones from each library were sequenced after 4 rounds of HER2-ECDselection. Sequence data was normalized to adjust for codons that wererepresented more than once. In most libraries there were few siblings(clones with identical DNA sequence). However, library-4 was dominatedby a single sequence with only 7 unique sequences total, and since allbut two residues in library-4 were mutated elsewhere, it was omittedfrom further analysis. Any siblings in the remaining libraries were alsoomitted in the analysis of amino acid variability.

Using this measure, the variability of phage selected amino acids couldbe compared to the natural variability of roughly 2000 human Ig κ lightchains and 4500 human Ig heavy chains found in the Kabat database(Johnson, G. & Wu, T. T., Nucleic Acids Res 29(1):205-6 (2001)). Theresults (FIG. 5) showed extremely diminished variability in the hu4D5-8residues as compared with variability of a wide range of antibodies inthe Kabat database. However, within the hu4D5-8 binding site, there areclearly differences in variability of the residues examined here. Thesepositions were ranked according to their variability score: Class 1,relatively invariant residues (V_(S)<10); Class 2, moderately variableresidues (V_(S)=10 to 40); and Class 3, highly variable residues(V_(S)>40). Sequence information for clones selected from the fourlibraries is shown in FIG. 3, presented according to this classificationsystem. All amino acids were observed at some frequency and position,but Cys and Gln were only rarely observed (2-3% at two positions each).Also studied was the relationship between the patterns of variabilityand the effects of substitutions on binding affinity.

Overall, wild-type residues were strongly conserved at many positions.Heavy-chain residues Y33, R50, Y56, R58, W95, G99, F100, and Y100a alongwith light-chain residues F53 and T94, were all conserved withnormalized frequencies ≥45% (FIG. 3). Y100a(V_(H)), a>12,000-fold effectwhen mutated to Ala (Kelley, R. F. & O'Connell, M. P., Biochemistry32(27):6828-35 (1993)), R50(V_(H)), a>2000-fold effect when mutated toAla, and G99(V_(H)), not previously mutated, were about 90-100%conserved—the latter two in two independent libraries. On the otherhand, some of the residues appearing in multiple libraries did showcontext-dependent differences in amino acid occurrences. W95(V_(H)),a>18,000-fold Ala hit, showed wild-type as the preferred residue with82% frequency in one library (library 2), but with only 59% in another(library 1). F100(V_(H)), a modest 7-fold Ala hit, was rather stronglyconserved in one library (library 3), but approximately equally oftensubstituted by Trp or Met in another (library 5). F53 (V_(L)) is anotherexample of how some selected residues varied with context. In library 3,the wild-type Phe was preferred by 67% to 16% over Trp, while in library5, the preference was reversed with Trp favored over Phe by 55% to 16%(FIG. 3).

Wild-type residues were not so predominant at other positions.Interestingly, at T94(V_(L)), where Ala substitution had little effect,there was a 45% conservation of Thr, but also a rather high occurrence(27%) of a chemically distinct substitution, Trp. Several light-chainresidues that had shown a range of Ala-scan effects from 6-fold to200-fold also showed strong selection (≥45% frequency) of non-wild-typeresidues, but preserved the wild-type chemical character: N30(V_(L)),Y55(V_(L)), H91(V_(L)) in two libraries, and Y92(V_(L)). At V_(L)30 thewild-type Asn occurred in only 34% of the selected clones and wasreplaced with Ser in 53% of the selected clones. Interestingly, at theneighboring V_(L)92, Trp occurred in 41% of the selected clones, whileMet, Phe, and Tyr (wild-type) each occurred in 16-19% of the clones. TheTyr at V_(L)55 was preferably substituted by Trp (58%) and lessfrequently by Phe or Tyr (12% each). Because these types of substitutionin general had unpredictable effects on binding affinity, they wereexamined further (see below) as point mutations in the context ofsoluble hu4D5-8 Fab preparations.

At the remaining three positions, Y49(V_(L)), D98(V_(H)), andY102(V_(H)), the pattern of substitutions were more complex, with nosingle amino acid occurring with more than about 30% frequency. Theseresidues retained WT identity with frequency <10% and had the largestvariety of amino acids. Y49(V_(L)) (along with Y55(V_(L)) faced V_(H)100and V_(H)102 at the light-chain:heavy-chain interface. V_(H)49 waspoorly conserved (only 9% WT) in two libraries and showed a preferencefor Trp or Phe, depending on the context. The Tyr at V_(H)102 was a sidechain from the murine CDR that was added during humanization (Carter etal., Proc Natl Acad Sci USA 89(10):4285-9 (1992b)) to improve bindingaffinity; however, the point mutation in isolation did not affectaffinity (Kelley, R. F. & O'Connell, M. P., Biochemistry 32(27):6828-35(1993)). From the phage library data, the human framework residue, Valwas actually preferred at this position. Both Val and Tyr often occur inhuman Ig heavy chains at this location (Johnson, G. & Wu, T. T., NucleicAcids Res 29(1):205-6 (2001)). A neighboring residue, V_(L)55, waspoorly conserved (mentioned above). The most poorly conserved residuewas V_(H)98. D98(V_(H)) is located in the tip of variable loop 3 of theheavy chain. It was mutated in 98% of all selected clones andsubstitution of every amino acid except Cys was observed there. The mostfrequent amino acid at this position was Trp, occurring with 23%frequency. These substitutions were also of particular interest, andthey are examined further (see below) as point mutations in the contextof soluble hu4D5-8 Fab preparations.

Comparison of Sequence Variability and Ala Scan Data

An alanine scan replaces WT residues with alanine and generally measuresloss of function. A goal for the phage libraries used in theseexperiments was to maintain function through random substitutions andantigen-binding selection. The degree of sequence variation using theV_(S) parameter (FIG. 5) with the Ala-scan map (Kelley, R. F. &O'Connell, M. P., Biochemistry 32(27):6828-35 (1993)) of hu4D5-8 in thecontext of the crystal structure of the antibody (FIG. 7) were compared.The two maps showed some common and some distinct features.

Four residues of hu4D5-8 had been found to be most critical for antigenbinding in the binding site of HER2-ECD based on alanine mutagenesis(Kelley, R. F. & O'Connell, M. P., Biochemistry 32(27):6828-35 (1993)).The importance of three of these residues (see FIG. 7), R50(V_(H)),W95(V_(H)), and Y100a(V_(H)), was confirmed in that they wereconsistently selected as their WT identity despite the context in whichthey were randomized. However, phage display also selected fouradditional residues (N30(V_(L)), R56(V_(H)), R58(V_(H)), and G99(V_(H)))that are highly conserved as WT that were not detected by alaninescanning (FIG. 7). Two of these, N30(V_(L)) and R56(V_(H)), were foundto decrease K_(D) by 4 to 6-fold when mutated to alanine.

One discrepancy between the alanine scan and phage display results wasV_(L)91. When V_(L)91 was mutated to alanine there was a 200-folddecrease in binding. V_(L)91 was mutated to a Phe in 44-45% of allselected clones in two different libraries. WT hu4D5-8 has a His in thisposition. While this His has only 22% exposed surface area in thecrystal structure there was still room to fit a Phe (Eigenbrot et al., JMol Biol 229(4):969-95 (1993)). The extra aromatic ring could packagainst nearby residues and extend the hydrophobic core.

Ala occurred in only 6 (V_(L)49, V_(L)53, V_(L)55, V_(H)98, V_(H)99,V_(H)102) of the 19 residues that were randomized and it at a frequencyof <6% for five of these residues, 14% for the other (V_(H)99). Four ofthese six residues were included in the alanine scan and all four ofthem were shown to decrease K_(D) by 2-fold (Kelley et al., Biochemistry31(24):5434-41 (1992)). The lack of alanine selection at these positionsagreed with these results, provided that the HER2 binding selectionunder the conditions employed here was generally efficient ineliminating variants with >2-fold reductions in binding affinity.Analysis of Fab binding affinities of prevalent substitutions supportedthis (FIGS. 2 and 3) because all of the high-frequency (>50%) variantsshowed binding affinities within 2-fold of wild-type. In contrast, alow-frequency (10%) substitution, Y100aF(V_(H)), demonstrated about5-fold weaker binding affinity than that of hu4D5-8 (FIG. 4).

The phage library selection was intended to select mutants with highaffinity for antigen. Conservation of particular side chains could bethe result of direct antigen contact, requirements for antibodystructural stability or expression, or a combination of these effects.An example of a likely structure-stabilizing conservation was atG99(V_(H)). V_(H)99 was a highly conserved residue in variable loop 3 ofthe heavy chain. This residue is position i+2 loop is a type II β-turnwhich is most commonly a Gly (Wilmot, C. M. & Thornton, J. M., J MolBiol 203(1), 221-32 (1988)). Therefore, it seemed likely to be astructure-stabilizing residue.

Chemical Characteristics of Observed Substitutions

Aliphatic side chains were not specifically targeted in the librariesexamined here. Perhaps not surprisingly, hydrophobic substitutionsfailed to dominate at any given site. However, there were a large numberof hydrophobic residues that appeared at low levels in the mutatedclones. Of these, the highest occurrence was only 23% for Leusubstituted at R58(V_(H)).

Two polar side chains were well conserved: R50(VH) and R59(VH). However,H91(VL) and D98(VH) were more often substituted with a nonpolar aromaticresidue, and T94(VL) was sometimes substituted with Trp. D98W(VH) wasparticularly interesting because it improved antigen binding asdiscussed below.

Conservation of chemical character occurred especially among aromaticand hydrophobic residues in the hu4D5-8 libraries. There are 6 residues(V_(H)100a, V_(H)56, V_(L)53, V_(H)33, V_(H)95, and V_(L)55) thatfavored an aromatic in 80% or more of the selected clones. Fivepositions (V_(L)49, V_(L)53, V_(L)91, V_(L)92, V_(H)100) selectedaromatics 50% or more of the time. While there was often some biastowards one, in one case, V_(H)100, a Phe and a Trp occurred equallyoften (30% or 34%).

Three highly conserved residues were likely involved in a cation-πinteraction. This interaction was between an Arg (V_(H)50), a Tyr(V_(H)33), and a Trp (V_(H)95) (Gallivan, J. P. & Dougherty, D. A., ProcNatl Acad Sci USA 96(17):9459-64 (1999)). When mutated in one library,Arg occurred at V_(H)50 in 93% of the clones. Trp V_(H)95 was selectedin 59% of the clones and Tyr V_(H)33 in 61% of selected clones. Otheramino acid substitutions at V_(H)95 and V_(H)33 were by otherhydrophobic residues like Phe or Tyr (V_(H)95) and Trp (V_(H)33). Thesemutations were all likely to preserve the stabilizing chemistry with ArgV_(H)50. This result was supported by the fact that two of theseresidues, V_(H)50 and V_(H)95, drastically increased ΔΔG when mutated toalanine (Kelley, R. F. & O'Connell, M. P., Biochemistry 32(27):6828-35(1993)).

Other aromatics were also conserved. On the surface of the hu4D5-8binding site these surrounded a region of highly conserved residues(FIG. 7). Several of these mutations involved a set of putative π-πinteractions. One example was at positions V_(L)53, V_(L)49 andV_(H)100. In hu4D5-8 these were a phenylalanine, a tyrosine, and aphenylalanine, respectively. The structure of hu4D5-8 illustrated thatthese residues were within 5 Å of each other and stacked the aromaticrings face to face or in the preferred T-shaped conformation typical ofa π-π interaction (Burley, S. K. & Petsko, G. A., Science 229(4708):23-8(1985)). In the context of library 3 the outer phenylalanines wereconserved while the Tyr in the middle preferred Phe in 28% of theselected clones. In a different library, that of library 5, all threeresidues were altered. At V_(L)49, 31% of the clones had a Trp while 55%had a Trp at V_(L)53 and 64% had either Trp or Phe at V_(L)100. The factthat these positions preferred aromatic amino acids in both of thelibraries in which they were mutagenized suggested that conservation ofthe stabilizing π-π interaction on the surface of hu4D5-8 was importantto antibody structure. They may have also contributed to antigen bindingcontacts.

Another n-n interaction appeared to occur between two tyrosines (V_(L)55and V_(H)102) located at the interface of the light and heavy chain.These tyrosines were in a T-shaped geometry and could be a source ofstabilization. Surprisingly, this interaction was lost in the selectedclones. Thirty-five percent of the clones replace V_(L)55 with Trp whileV_(H)102 had a 19% occurrence of valine. Almost any other amino acidcould occur at V_(H)102, but valine was slightly preferred.

Non-Additive Effects on Binding Free Energy

The hu4D5-8 phage libraries distributed 19 surface residues among 5libraries. Several residues were present in more than one library toallow those in proximity to covary. Of the residues represented induplicate libraries, some differences were observed based on context asnoted above. However, based on a statistical analysis of covariation,there were no significant pair-wise correlations of substitutions at anyof these positions, although a much larger number of sequences mightmake these correlations more apparent.

While most of the Fab mutants had slight negative effects on K_(D), thecombination of all tested point mutations in the multiple mutant M.7still gave an improved binding affinity. Several single point mutationswere clearly not additive in hu4D5-8 as mutations such as Y100aF(V_(H))and Y92W(V_(L)) that adversely affected binding and/or folding stabilitywere “rescued” by combinations with other mutations to a greater extentthan would be predicted by additivity principles (Wells, J. A.,Biochemistry 29(37), 8509-17 (1990)).

Example 3 Hu4D5-8 Variant Fab Constructs

The assays discussed in Example 2 above demonstrated qualitatively thatall tested clones retained high affinity (nanomolar to sub-nanomolar)binding affinity to antigen. In addition to those assays, the bindingaffinity of soluble Fab fragments was also tested.

Selection of Clones for Fab Binding Experiments

Eight clones using point mutations in the context of soluble Fabfragments, representing high frequency of occurrence of non-wild-typeresidues and a range of variability scores, were selected and tested todetermine how selected substitutions affected HER2 binding as comparedwith the hu4D5-8 Fab.

Although siblings data were not used in analyzing the variety ofmutations at individual sites, these data were considered in designingvariants for binding experiments. It was reasoned that if a single clonewas present in multiple copies after several rounds of selection itcould potentially be due to improvements in binding, stability orexpression. Three of the most abundant siblings were chosen: a triplemutant, called M.3 (N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W) and the singlemutants T94(V_(L))S and Y100a(V_(L))F. These clones represented roughly20% of the total number of sequenced clones. All individual mutationswere also combined into one multiple-mutant clone, M.7,(N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W+T94(V_(L))S+D98(V_(H))W+Y100a(V_(H))F+Y102(V_(H))V).

Fab Constructs and Purification

Mutations were introduced with QuikChange® mutagenesis (Stratagene, LaJolla, Calif.) to Fab-expression plasmids pAK19 described previously(Carter et al., Proc Natl Acad Sci USA 89(10):4285-9 (1992b)). Hu4D5-8Fab mutants were overexpressed by secretion in E. coli (Carter et al.,Biotechnology (N Y) 10(2):163-7 (1992a)) and purified using a protein-Gaffinity column (Kelley et al., Biochemisty 31(24):5434-41 (1992)). Theconcentration of each mutant was determined spectrophotometrically aswell as by quantitative amino acid analysis; the two methods agreedwithin 5-12%.

Surface Plasmon Resonance (SPR) Binding Affinity Measurements

Surface plasmon resonance (SPR) was used to measure the binding kineticsof overexpressed Fabs to immobilized HER2-ECD receptor. A BIAcore-2000or BIAcore-3000 real-time kinetic interaction analysis system (BiacoreInc., Piscataway, N.J.) was used to determine association (k_(on)) anddissociation (k_(off)) constants (Karlsson et al., J Immunol Methods145(1-2):229-40 (1991)) of the hu4D5-8 Fab mutants. A B1 biosensor chip(Biacore, Inc.) was activated according to the manufacturer'sinstructions and immobilized with 86 to 500 RU's (response units) ofHER2-ECD in 10 mM sodium acetate, pH 4.8. Unreacted groups were blockedwith 1M ethanolamine. The kinetics of hu4D5-8 mutants binding toimmobilized HER2-ECD were measured with 2-fold serial dilutionsbeginning with 100 nM Fab in running buffer (PBS, 0.05% Tween, 0.01%sodium azide) at a flow rate of 20 μl/min. Binding measurements wererecorded at 19° C., 25° C., 31° C., and 37° C. at 4 different densitiesof immobilized HER2-ECD. Data were fit to a 1:1 Langmuir binding modelusing BIAcore evaluation software version 3 which calculated association(k_(on)) and dissociation (k_(off)) rates. An equilibrium constant,K_(D), was calculated from k_(off)/k_(on). Free energy differences, ascompared with wild-type hu4D5-8 were calculated as described (Wells, J.A., Biochemistry 29(37), 8509-17 (1990)): ΔΔG=−RT ln (K_(D)^((mutant))/K_(D) ^((wild-type)).

Antigen Binding Affinities of Fab Variants

Kinetics data for mutant Fabs binding to HER2-ECD at physiologicaltemperature (37° C.) is shown in FIG. 4. The Fab mutants generally hadvery similar association and dissociation rate constants, k_(on) andk_(off). As a result, most of the mutants had K_(D)'s similar to hu4D5-8Fab. One mutant, Y100a(V_(H))F, had a 4-fold negative affect on K_(D).Two mutants had improved K_(D)'S, the multi-mutant M.7,(N30(V_(L))S+H91(V_(L))F+Y92(V_(L))W+T94(V_(L))S+D98(V_(H))W+Y100a(V_(H))F+Y102(V_(H))V),by 1.5 fold and single mutant D98(V_(H))W by about 3-fold at 37° C.(FIG. 4).

Over a temperature range of 19°-37° C., all of the variants testedshowed binding energies well within 1.0 kcal/mol of that for binding ofthe wild-type hu4D5-8 to HER2 (FIG. 6). Over the same range, hu4D5-8affinity was essentially constant, with K_(D) ranging from 0.13 to 0.33nM.

Binding constants for mutant Y92(V_(L))W were not reported because itexpressed 10-fold more poorly than any of the other Fabs and showed poorbinding to HER2. While it was selected by phage, these results indicatethat Y92(V_(L))W was unable to function in the wild-type hu4D5-8background. Interestingly, this mutation was “rescued” in the context ofeither of the multiple mutants, M.3 or M.7. Fusion to the g3 protein mayassist in folding, as observed for certain phage displayed mutants ofIGF-1 which were poorly behaved as soluble proteins (Dubaquie, Y. &Lowman, H. B., Biochemistry 38(20):6386-96 (1999)).

Identification of an Affinity-Improved Variant

The binding affinity of hu4D5-8 Fab has been reported to be in thesub-nanomolar range (Kelley, R. F. & O'Connell, M. P., Biochemistry32(27):6828-35 (1993)). A single mutant. D98(V_(H))W, was selected ashaving a 3-fold improvement over WT. D98(V_(H)) is located at the tip ofvariable loop 3 of the heavy chain and is the most protruding residueson the surface of the antibody (Eigenbrot et al., J Mol Biol229(4):969-95 (1993)). Furthermore, it is adjacent to W95(V_(H)), one ofthe four strong hits in the alanine scan. D98(V_(H)) is the mostvariable position of all randomized 19 residues. Trp does not dominatethe selected pool, but is the most frequent substitution selected. Thelocation Trp V_(H)98 on the surface next to the putative binding sitesuggests this could be a site of sequence plasticity that directlycontacts antigen.

Example 4 Stringent Off-Rate Binding Selection Using 4D5 Fab-PhageLibraries

To search for additional high-affinity variants of humanized 4D5, thefive libraries of 4D5 variants described in the previous Examples (see,also Gerstner et al., J. Mol. Biol. 321, 851-862 (2002)) were used forbinding selections with immobilized HER2 as the binding target.Additional libraries were designed and constructed based upon theresults of selections using the initial libraries. In particular,libraries 6 and 7 were designed to target a combination of residuesidentified in the initial libraries with restricted diversity usingselected degenerate codons, and to include diversity at positionsproximal to those identified earlier. Table 2 summarizes the diversityengineered into these libraries.

TABLE 2 Design of 4D5 libraries 5 and 6. Library Chain Position CodonResidues Encoded Library-6 VL 30 ARC N, S VL 49 KKS F, L, W, V(2), C,G(2) VL 53 TKS W, F, L, C VL 55 TDS W, L, F, C, Q*, Y VL 91 YWC F, H, L,Y VL 92 TDS W, L, F, C, Q*, Y VL 94 WCC S, T VH 100 WKS F, L, C, W, I, MVH 102 STC L, V Library-7 VL 27 NNK (all) VL 28 NNK (all) VL 30 ARC N, SVL 31 NNK (all) VL 32 NNK (all) VL 66 NNK (all) VL 91 YWC F, H, L, Y VL92 TDS W, L, F, C, Q*, Y

In the foregoing Table 2 positions are shown according to the numberingsystem of Kabat (Kabat et al., Sequences of Proteins of ImmunologicalInterest. 5^(th) Edition, National Institutes of Health, Bethesda, Md.(1991)). Degenerate codons are shown using IUPAC code (R=A/G, Y=C/T,D=A/G/T, K=G/T, S=G/C, W=G/T, N=A/G/C/T).

In this series of binding-selection experiments, 4D5-phage librarieswere propagated and subjected to sorting essentially as described(Lowman, Methods Mol. Biol. 87, 249-264 (1998); Chen et al., J. Mol.Biol. 293, 865-881 (1999)). Briefly, immunosorbant plates (NuncMaxisorp) were coated with 2 μg/mL HER2-ECD in PBS (phosphate bufferedsaline) and blocked with BSA (bovine serum albumin). Thereafter, phagewere added at a concentration of about 1011 phage/mL in PBS containingBSA and Tween-20.

For stringent off-rate selections, phage binding was allowed to reachequilibrium over a period of 16 hours or longer, followed by washingwith PBS/Tween-20, and dissociation in wash buffer containing 0.01%sodium azide (with or without rhuMAb 4D5 antibody) for progressivelylonger periods of time (Table 3). Phage were eluted with a brief (10min.) incubation with 100 mM HCl, neutralized, and propagated overnightin XL1-Blue cells (Stratagene) as described (Lowman, 1998, supra).

TABLE 3 Conditions for off-rate binding selections Binding Round timeWashes Dissoc. Time Dissoc. Buffer 1 48 h 10 x — — 2 16 h 20 x  3 h Washbuffer 3 16 h 10 x daily  48 h Wash buffer + 100 nM 4d5 4 16 h 10 xdaily 120 h Wash buffer + 100 nM 4d5

Binding enrichments were measured by comparison of recovered phagetiters from HER2 versus BSA wells. The results over increasinglystringent rounds of binding selection showed enrichment of HER2-ECDbinding phage over background binding for each library except library 4(data not shown).

Phage clones were isolated after four rounds of binding selection forsequencing and further characterization. The sequences of these clonesare shown in Table 4, in comparison with the wild-type (rhuMAb 4D5)residue at each position. A statistical test of significance (Lowman &Wells, J. Mol. Biol. 234, 564-578 (1993)) was applied to define favoredsubstitutions at each position where non-wild-type residues werecommonly observed (Table 4). Briefly, the observed frequency (Pobs) ofeach amino acid is compared to the expected (random) frequencydetermined from the number of codons that can encode that amino acid(Pexp), using a particular codon degeneracy. The significance score, S,is calculated as S=(Pobs-Pexp)/σ, where σ is the standard deviation ofthe theoretical random distribution (Lowan & Wells, 1993, supra).

TABLE 4 Sequences of 4d5-phage isolates after fourrounds of off-rate binding selection using immobilized HER2-ECD.Library 1 VL VH Position: 94 33 50 56 58 95 WT T Y R Y R W 4d5.26 T Y RA R W 4d5.29 T Y R Y R Y 4d5.32 T Y R Y R Y 4d5.34 T Y R Y R Y 4d5.37 TY 4d5.39 T Y 4d5.41 T W R Y R F 4d5.44 T F 4d5.45 T Y R A R W 4d5.35 T WR W I Y 4d5.36 T W R W I Y 4d5.27 T Y R Y R F 4d5.33 T Y R Y R W 4d5.30T Y R Y R Y 4d5.43 T Y 4d5.38 T Y R Y R Y 4d5.42 T F R Y R WConsensus changes: Y Significance score: 13 Library 2 VL VH Position: 3091 92 50 95 99 100a WT N H Y R W G Y 4d5.1 S F W 4d5.10 S F W R W G Y4d5.12 S F W R W G Y 4d5.7 S F W 4d5.3 S Y W R W G Y 4d5.8 S F W R W G Y4d5.11 S W R W G Y 4d5.9 S F F R W G Y 4d5.2 S F G R W G Y 4d5.4 S Y G RW G Y 4d5.5 S I W R W G Y 4d5.6 S F W R W G Y Consensus changes: S F WSignificance score: 11 13 14 Library 3 VL VH Position: 49 53 91 98 99100 100a WT Y F H D G F Y 4d5.15 W V Y H G M Y 4d5.22 W F Y A G F N4d5.16 L F H R S Y Y 4d5.24 F F Y A S L F 4d5.21 W V F R G L Y 4d5.23 SW F S G F Y 4d5.18 K F Y T G A Y 4d5.19 Y F F K G F Y 4d5.13 V W Y4d5.14 V F F 4d5.17 F W Y L G H Y 4d5.20 L V H L Y Y **4d5.31 L L T4d5.28 W W W V Consensus changes: W W Y/F basic Significance score: 5.55.5 8.9/5.7 3.0 Library 5 VL VH Position: 49 53 55 100 102 WT Y F Y F Y4d5.50 D W W P K 4d5.51 D W W P L 4d5.52 D W W P L 4d5.54 V T W P W4d5.53 Y F W 4d5.59 F H W W M 4d5.57 V V W W L 4d5.60 A V L H L 4d5.49 WR W 4d5.55 W Q F F W 4d5.56 V W L P H 4d5.58 W T Y F Y 4d5.89 D W W4d5.92 D W W P K 4d5.93 D W W P L 4d5.94 D W W 4d5.77 Y F W P K 4d5.86 FM W 4d5.85 E W W 4d5.96 R W V 4d5.87 T K W 4d5.91 R A W 4d5.88 T R V4d5.90 V K S M A 4d5.95 S V W Consensus changes: D W W PSignificance score: 7.1 11 20 7.9 Library 6 (restricted) VL VH Position:30 49 53 55 91 92 94 100 102 WT N Y F Y H Y T F Y 4d5.63 N V W W H Y T4d5.64 L S 4d5.65 N V W W H Y T L S 4d5.66 N V W W H Y T 4d5.68 N V W WH Y T 4d5.70 N V W W H Y T L S 4d5.61 N F K W H Y T L T 4d5.71 N V W W HY T 4d5.67 N V R A H Y T M G 4d5.69 N V R A H Y T M G 4d5.62 N W L P H YT M 4d5.72 N L M G H Y T R L Consensus changes: V W WSignificance score: 6 2.3 4.2 Library 7 (restricted) VL VH Position: 2728 30 31 32 66 91 92 100 102 WT Q D N T A R H Y F Y 4d5.73 S Q* S S G RH W 4d5.76 S Q* S G G R H W P A 4d5.75 R Q* N T A R F F 4d5.83 A Q* S AG R Y W P V 4d5.79 Q* G S S G A H W 4d5.80 Q* G S S A N H W P K 4d5.78 QR N S A R H F 4d5.81 Q* G S S A M H F P L 4d5.74 N P S Q A T H W 4d5.84S Q* S K A S Y L P L 4d5.82 F N A C V H Q* P L Consensus changes: Q S GW P Significance score: 5.7 4.1 4.1 3.4 5.5

No sequencable clones were recovered from round 4 of selections usingLibrary 4. In the foregoing table Gln residues encoded by read-throughof the amber stop codon (TAG) are indicated by Q*. A spontaneousmutation (V_(H) Y102M) was identified at a site not targeted formutagenesis in the original libraries (**). Consensus residues are shownfor positions where non-wild-type residues occurred with significantfrequency. The restricted codon selections used in libraries 6-7 aredescribed in Table 2. In Library 3, “basic” residues refer to acombination of H, K, and R. Blanks indicate uncertain or undeterminedsequence at the corresponding position.

The occurrence of non-wild-type residues may reflect improved bindingaffinity, stability, and/or expression level for variants containingthose substitutions. However, in previous affinity maturation studies,significance scores >2 have often correlated with improvements inbinding affinity (Lowman & Wells, 1993, supra). Therefore, based uponthe sequence-significance scores from Table 4, substitutions that mayimprove the binding affinity of 4d5 for HER2 are listed in Table 5.These substitutions can be compared with the finding of Gerstner et al.(2002), supra. For example, in that work, using solution-phase captureof 4D5-phage, several of the substitutions identified here were alsofound. Some positions showed similar substitutions, for example, VLmutations F53W, Y55W, and Y92W, were commonly found in the previousexperiments. However, substitutions not commonly found in the previousexperiments include basic residues (R, K, H) substituting at VH position98, and P substituting at VH position 100. These mutations may actindividually, or in combination with other mutations to improve bindingaffinity of 4D5 for HER2.

TABLE 5 Summary of consensus residues by position from off-rateselections. Chain Position Preferred residue(s) VL D28 Q VL N30 S VL T31S VL A32 G VL Y49 W, D, V VL F53 W VL Y55 W VL H91 Y, F VL Y92 W VH W95Y VH D98 R, K, H VH F100 P

Example 5 Screening of Selected 4D5 Clones from Off-Rate Selection

Selected representative clones were chosen for further characterizationin competitive phage-ELISA assays (Lowman, 1998). Several variantsappeared to have improved binding to HER2-ECD as compared with wild-type4D5 Fab (Table 6). Because of the relatively high affinity of wild-type4D5, this assay does not provide a reliable measure of affinity-maturedversions of 4D5 (Gerstner et al., 2002); however, we have used the assayto rank clones for further analysis.

TABLE 6 Competitive phage-ELISA results for selected 4d5-phage. RelativeClone IC₅₀ s.d. WT -1- — 4d5.2 0.15 0.11 4d5.4 0.21 0.15 4d5.17 0.090.07 4d5.21 1.98 1.41 4d5.22 0.06 0.04 4d5.28 0.10 0.07 4d5.31 0.18 1.064d5.35 0.35 1.04 4d5.44 0.40 0.82 4d5.50 1.49 0.85 4d5.51 1.47 0.284d5.55 1.16 0.04 4d5.57 1.20 0.85 4d5.64 0.40 0.28 4d5.67 0.05 0.044d5.80 1.93 1.37 4d5.81 1.26 0.89 4d5.83 0.08 0.06 4d5.84 0.21 0.144d5.92 2.08 0.67 D98W.1 2.76 1.13 D98W.2 3.77 0.95

The relative IC50 reported is calculated asIC₅₀(wild-type)/IC₅₀(variant); values >1 reflect higher apparentaffinities than wild-type. Errors are reported as standard deviations(s.d.). For comparison, values for two independent clones of apreviously reported variant, D98W (VH) are also shown.

Based on the results of phage-ELISA assays, several variants werepredicted to have improved binding affinity to HER2: 4d5.21, 4d5.50,4d5.51, 4d5.55, 4d5.57, 4d5.80, 4d5.81, and 4d5.92. The point mutationsidentified among all these clones are summarized in Table 7. Thesemutations are therefore implicated as acting separately orsynergistically to improve binding affinity of 4d5 to HER2.

TABLE 7 Summary of point mutations by position found among highestaffinity variants identified by phage-ELISA screening. Chain PositionPreferred residue(s) VL D28 G VL N30 S VL T31 S VL Y49 W*, D*, V VL F53V*, W*, Q VL R66 N, M VL H91 F*, W* VL Y92 W, F VH D98 R*, W VH F100 P*,L*, W VH Y102 W, L, K* Mutations occurring in the two highestapparent-affinity variants, 4d5.21 and 4d5.92, are indicated (*).

Example 6 Affinity Measurements of Selected 4D5 Clones Fromo Off-RateSelection

To determine equilibrium binding affinities (Kd) of Fab variants,soluble Fab fragments produced in E. coli and tested in a BIAcorebinding assay (Gerstner et al, 2002) using immobilized HER2-ECD at 37°C. Fab concentrations were determined by quantitative amino acidanalysis. The results of kinetic measurements are summarized in Table 8.

TABLE 8 Binding kinetics and affinities of selected 4d5 Fab variantsusing a surface-plasmon resonance assay (BIAcore). k_(on) k_(off)Relative Variant (/10⁶/M/s) (10⁴/s) K_(d) (pM) s.d. n Affinity 4d5.511.32 0.18 14  8 2 7.6 4D5.80 1.49 0.23 16 n/a 1 6.7 D98W 2.99 0.80 27 1512 3.9 4d5.50 1.94 0.81 42 25 3 2.5 4d5.21 3.57 2.60 73 28 9 1.4 WT 1.902.01 105 29 12 1.0 4D5.55 1.56 4.40 281 72 10 0.4

In the foregoing Table 8 equilibrium dissociation affinities (Kd) arecalculated as koff/kon, for n measurements. Errors are shown as standarddeviations (s.d.). The relative affinity is calculated asKd(WT)/Kd(variant); values >1 indicate higher apparent affinity forHER2-ECD.

The results of these experiments indicate that Fabs corresponding tophage clones 4d5.51, 4d5.80, and 4d5.50, as well as the previouslydescribed point mutant D98W (VH) each have >2-fold improved binding toHER2-ECD as compared with WT. For comparison, the substitutions found ineach of these variants are summarized in Table 9. On the other hand,4D5.21 and 4D5.55 have little improvement, or are slightly weaker inbinding.

Comparison of relative on-rates and off-rates indicates that while D98W,identified by solution-phase binding selections (Gerstner et al, 2002)was improved in both kon and koff, the best variants identified bystringent off-rate selections using immobilized 4d5 consistently hadslower koff, with slower kon as compared with WT.

TABLE 9 Point mutation in affinity-improved    4d5 variants identified by kinetics    analysis. Residues differing from   WT are shown in bold; residues identical to WT are shown (-). Variant Posi- VL VL VL VL VL VL VL VL VLVL VL VH VH VH tion: 27 28 30 31 32 49 53 55 66 91 92 98 100 102 WT Q DN T A Y F Y R H Y D F Y 4d5.50 - - - - - D W W - - - - P K4d5.51 - - - - - D W W - - - - P L 4d5.80 - G S S - - - - N - W - P KD98W - - - - - - - - - - - W - -

Example 7 4D5 Variants Produced by Combinations of Selected Mutations

Because mutations could act individually or in combination with othermutations found in the same 4d5-phage selectant, we were interested intesting combinations of mutations from the highest affinity variants,including the previously described D98W (VH). A set of variants weredesigned to test the contributions of “DWW” (i.e., VL mutationsD49D/F53W/Y55W) alone, as well as “DWW” and “PL” or “PK” (i.e., VHmutations F100P/Y102K or F100P/Y102L) in combination with the VHmutations D98W (Table 10).

TABLE 10 Combination variants of 4d5. Variant Posi- VL VL VL VL VL VL VLVL VL VL VL VH VH VH tion: 27 28 30 31 32 49 53 55 66 91 92 98 100 102WT Q D N T A Y F Y R H Y D F Y 4d5- - - - - - - - - - - - W P K D98W- PK4d5- - - - - - - - - - - - W P L D98W- PL 4d5- - - - - - D WW - - - - - - DWW 4d5- - - - - - D W W - - - W - - D98W- DWW

Variant Fabs were produced by site-directed mutagenesis, expressed in E.coli, and assayed by BIAcore binding at 37° C. In these assays,association and dissociation constants were measured as previouslydescribed (Gerstner et al., 2002), except that the dissociation phase ofeach experiment was extended to 30 min. to permit more accuratemeasurement of the very slow koff rates observed, Results are shown inTable 11.

TABLE 11 Binding affinities from kinetics analysis (BIAcore) of 4d5variants combining mutations from selected 4d5-phage variants. k_(on)k_(off) Relative Variant (/10⁶/M/s) (10⁴/s) K_(d) (nM) s.d. n AffinityWT 0.69 2.19 317 87 6 1.00 D98W 1.26 0.95  75 27 6 4.23 4d5.50 0.87 0.87111 50 3 2.86 4d5.51 0.67 0.53  69 17 3 4.62 4d5.80 N.D. N.D. N.D. N.D.N.D. N.D. 4d5-D98W-PK 0.44 5.04 1146  109  3 0.28 4d5-D98W-PL 0.50 4.29856 141  3 0.37 4d5-DWW N.D. N.D. N.D. N.D. N.D. N.D. 4d5-PK N.D. N.D.N.D. N.D. N.D. N.D. 4d5-D98W- N.D. N.D. N.D. N.D. N.D. N.D. DWW

In the foregoing table, Kon and koff were fit separately using theBIAcore BIAevaluation software. Equilibrium dissociation affinities Kd)are calculated as koff/kon, for n measurements. Errors are shown asstandard deviations (s.d.). The relative affinity is calculated asKd(WT)/Kd(variant); values >1 indicate higher apparent affinity forHER2-ECD, N.D.=not determined.

The results of these long-dissociation assays confirmed that variantsD98W, 4d5.50, and 4d5.51 have improved binding affinity to HER2 ascompared with wild-type. However, combinations of D98W with 100P/Y102Kor F100P/Y102L did not produce additive improvements in theseexperiments.

All references cited herein and throughout the specification are herebyexpressly incorporated by reference.

Deposit of Biological Material

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

-   -   Antibody Designation ATCC No. Deposit Date    -   4D5 ATCC CRL 10463 May 24, 1990

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of the deposit. The organisms will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC § 122 and the Commissioner's rules pursuant thereto (including37 CFR § 1.12 with particular reference to 886 OG 638).

In respect of those designations in which a European patent is sought, asample of the deposited microorganism will be made available until thepublication of the mention of the grant of the European patent or untilthe date on which the application has been refused or withdrawn or isdeemed to be withdrawn, only by the issue of such a sample to an expertnominated by the person requesting the sample. (Rule 28(4) EPC).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiments are intended to illustrate only certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. The examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture should not be construed to limit the invention.

1. A polypeptide which comprises an antibody light chain variable domaincomprising the hypervariable regions of SEQ ID NO: 1 wherein one or moreamino acids selected from the group consisting of Q27(V_(L));D28(V_(L)), N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)),Y55(V_(L)), R66(V_(L)), H91(V_(L)), Y92 (V_(L)), and T94(V_(L)),numbered according to the Kabat numbering system, are substituted withany amino acid other than alanine.
 2. The polypeptide of claim 1,wherein. the hypervariable regions of SEQ ID NO: 1 comprise amino acidsubstitutions at one or more positions selected from the groupconsisting of N30(V_(L)), F53(V_(L)), Y55(V_(L)), H91(V_(L)),Y92(V_(L)), and T94(V_(L),).
 3. The polypeptide of claim I wherein thehypervariable regions of SEQ ID NO: 1 comprise one or more amino acidsubstitutions selected from the group consisting of D28(V_(L))Q;D28(V_(L))G; N30(V_(L))S; T31(V_(L))S; A32(V_(L))G; Y49(V_(L))W,Y49(V_(L))D, 49(V_(L))V; F53(V_(L))W. F53(V_(L))V, F53(V_(L))Q,Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F, H91(V_(L))Y, Y92(V_(L))W, andT94(V_(L))S.
 4. The polypeptide of claim 3 wherein the hypervariableregions of SEQ ID NO: 1 comprise one or more amino acid substitutionsselected from the group consisting of D28(V_(L))Q; D28(V_(L))G;N30(V_(L))S; T3I(V_(L))S; A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D,Y49(V_(L))V; F53(V_(L))W, F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W,R66(V_(L))N, H9I(V_(L))F, H91(V_(L))Y, and Y92(V_(L))W.
 5. Thepolypeptide of claim 4 wherein the hypervariable regions of SEQ ID NO: Icomprise one or more amino acid substitutions selected from the groupconsisting of Y49(V_(L))D, F53(V_(L))W, and Y55(V_(L))W.
 6. Thepolypeptide of claim 5 comprising two or three or said amino acidsubstitutions.
 7. The polypeptide of claim 6 comprising all three ofsaid amino acid substitutions.
 8. The polypeptide of claim 3, whereinthe hypervariable regions of SEQ ID NO: 1 comprise one or more aminoacid substitutions selected from the group consisting of N30(V_(L))S,F53(V_(L))W, Y55(V_(L))W, H91(V_(L)) F, Y92(V_(L))W and T94(V_(L))S. 9.The polypeptide of claim 8, wherein N30(V_(L)) substituted with S,H91(V_(L)), is substituted with F, and Y92(V_(L)) is substituted with W.10. The polypeptide of claim 1 which is an antibody.
 11. The polypeptideof claim 10, which is a humanized antibody.
 12. The polypeptide of claim10, which is a human antibody.
 13. The polypeptide of claim 10, which isan antibody fragment selected from the group consisting of Fv, Fab, Fab′and F(ab′)₂ fragments.
 14. A polypeptide which comprises an antibodyheavy chain variable domain comprising the hypervariable regions of SEQID NO: 2 wherein one or more amino acids selected from the groupconsisting of W95(V_(II)), D98(V_(II)), F100(V_(II)), Y100a(V_(II)), andY102(V_(H)), numbered according to the Kabat numbering system, aresubstituted with any amino acid other than alanine.
 15. The polypeptideof claim 14, wherein the hypes-variable regions of SEQ ID NO: 2 compriseone or more amino acid substitutions selected from the group consistingof W95(V_(H))Y, D98(V_(H))W, D98(V_(H))R, D98(V_(H))K, D98(V_(H))H,F100(V_(H))P, F100(V_(H))L, F100(V_(H))M, Y100(V_(H))F, Y102(V_(H))V,Y102(V_(H))K, and Y102(V_(H))L.
 16. The polypeptide of claim 15 whereinthe hypervariable regions of SEQ ID NO: 2 comprise one or more aminoacid substitutions selected from the group consisting of, D98(V_(H))W,Y100a(V_(H))F, and Y102(V_(H))V.
 17. The polypeptide of claim 15 whereinthe hypervariable regions of SEQ ID NO: 2 comprise one or two amino acidsubstitutions selected from the group consisting of F100(V_(H))P andY102(V_(H)) K.
 18. The polypeptide of claim 17 comprising the amino acidsubstitutions F100(V_(H))P and Y102(V_(H))K.
 19. The polypeptide ofclaim 15 wherein the hypervariable regions of SEQ ID NO: 2 comprise oneor two amino acid substitutions selected from the group consisting ofFI00(V_(H))P and V102(V_(H))L.
 20. The polypeptide of claim 19comprising the amino acid substitutions of F100(V_(H))P andY102(V_(H))L.
 21. The polypeptide of claim 14, which is an antibody. 22.The polypeptide claim 21, which is a humanized antibody,
 23. Thepolypeptide of claim 21, which is a human antibody.
 24. The polypeptideof claim 21, which is an antibody fragment selected from the groupconsisting of Fv, Fab, F(ab′)₂ and F(ab′)₂ fragments.
 25. An antibodythat is capable of binding to the extracellular domain of HER2 whichcomprises the hypervariable regions of SEQ ID NO: 1 wherein one or moreamino acids selected from the group consisting of Q27(V_(L)),D28(V_(L)), N30(V_(L)), T31(V_(L)), A32(V_(L)), Y49(V_(L)), F53(V_(L)),F55(V_(L)), R66(V_(L)), H91(V_(L)) and T94(V_(L)), numbered according tothe Kabat numbering system, are substituted with any amino acid otherthan alanine.
 26. The antibody of claim 25 wherein one or more aminoacids selected from the group consisting of N30(V_(L)), F53(V_(L)),Y55(V_(L)), H91(V_(L)), Y92(V_(L)), and T94(V_(L)), numbered accordingto the Kabat numbering system, are substituted with any amino acid otherthan alanine.
 27. The antibody of claim 25 wherein the hypervariableregions of SEQ ID NO: 1 comprise one or more amino acid substitutionsselected from the group consisting of D28(V_(L))Q; D28(V_(L))G;N30(V_(L))S; T3I(V_(L))S; A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D,Y49(V_(L))V; F53(V_(L))W, F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W,R66(V_(L))N, H91(V_(L))F, H91(V_(L))Y, Y92(V_(L))W, and T94(V_(L))S. 28.The antibody of claim 27 wherein the hypervariable regions of SEQ ID NO:1 comprise one or more amino acid substitutions selected from the groupconsisting of D28(V_(L))Q; D28(V_(L))G; N30(V_(L))S; T31(V_(L))S;A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D, Y49(V_(L))V; F53(V_(L)) W,F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W, R66(V_(L))N, H91(V_(L))F,H91(V_(L))Y, and Y92(V_(L))W.
 29. The antibody of claim 28 wherein thehypervariable regions of SEQ ID NO: 1 comprise one or more amino acidsubstitutions selected from the group consisting of Y49(V_(L))D,F53(V_(L))W, and Y55(V_(L))W.
 30. The antibody of claim 29 comprisingtwo or three or said amino acid substitutions.
 31. The antibody of claim30 comprising all three of said amino acid substitutions.
 32. Theantibody of claim 26, wherein the hypervariable regions of SEQ ID NO: 1comprise one or more amino acid substitutions selected from the groupconsisting of N30(V_(L))S, F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F,Y92(V_(L))W, T94(V_(L))S.
 33. The antibody of claim 32, whereinN30(V_(I)) is substituted with S, H91(V_(I)) is substituted with F, andY92(V_(L)) is substituted with W.
 34. The antibody of claim 25, which isa humanized antibody.
 35. The antibody of claim 25, which is a humanantibody.
 36. The antibody of claim 25, which is a antibody fragmentselected from the group consisting of Fv, Fab, Fab′ and F(ab′)₂fragments.
 37. An antibody that is capable of binding to theextracellular domain of HER2, which comprises an antibody heavy chainvariable domain comprising the hypervariable regions of SEQ ID NO: 2wherein one or more amino acids selected from the group consisting ofW95(V_(H)), D98(V_(H)), F100(V_(H)), Y100a(V_(H)), and Y102(V_(H)),numbered according to the Kabat numbering system, are substituted withany amino acid other than alanine.
 38. The antibody of claim 37 whereinthe hypervariable regions of SEQ ID NO: 2 comprise one or more aminoacid substitutions selected from the group consisting of W95(V_(H))Y,D98(V_(H))W, D98(V_(H))R, D98(V_(H))K, D98(V_(H))H, FI00(V_(H))P,F100(V_(H))L, F100(V_(H))M, 100a(V_(H))F, Y102(V_(H))V, Y102(V_(H))K,and Y102(V_(H))L.
 39. The antibody of claim 38 wherein the hypervariableregions of SEQ ID NO: 2 comprise one or more amino acid substitutionsselected from the group consisting of D98(V_(H))W, Y100a(V_(H))F, andY102(V_(H))V.
 40. The antibody of claim 38 wherein the hypervariableregions of SEQ ID NO: 2 comprise one or more amino acid substitutionsselected from the group consisting of F100(V_(H))P and Y102(V_(H))K. 41.The polypeptide of claim 40 comprising the amino acid substitutionsF100(V_(II))P and Y102(V_(II))K.
 42. The antibody of claim 20, which isa humanized antibody.
 43. The antibody of claim 37, which is a humanantibody.
 44. The antibody of claim 37, which is an antibody fragmentselected from the group consisting of Fv, Fab, Fab′ and F(ab′)₂fragments.
 45. An antibody that is capable of binding to theextracellular domain of HER2, which comprises the hypervariable regionsof SEQ ID NOs: 1 and 2 wherein one or more amino acids selected from thegroup consisting of D28(V_(L)), N30(V_(L)), T31(V_(L)), A32(V_(L)),Y49(V_(L)), F53(V_(L)), Y55(V_(L)), R66(V_(L)), H91(V_(L)), Y92(V_(L)),T94(V_(L)), W95(V_(H)), D98(V_(H)), F100(V_(H)); Y100a(V_(H)), andY102(V_(H)), numbered according to the Rabat numbering system, aresubstituted with any amino acid other than alanine.
 46. The antibody ofclaim 45, wherein the hypervariable regions of SEQ ID NOs: 1 and 2comprise one or more amino acid substitutions selected from the groupconsisting of N30(V_(L))S, F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F,Y92(V_(L))W, T94(V_(L))S, D98(V_(H))W, Y100a(V_(H))F, and Y102(V_(H))V.47. The antibody of claim 46, wherein N30(V_(L)) is substituted with S,H91(V_(L)) is substituted with F, Y92(V_(L)) is substituted with W,T94(V_(L)) is substituted with S, D98(V_(H)) is substituted with W,Y100a(V_(H)) is substituted with F, and Y102(V_(H)) is substituted withV.
 48. The antibody of claim 46, wherein D98(V_(H)) is substituted withW.
 49. The antibody of claim 45, wherein the hypervariable regions ofSEQ ID NOs 1 and 2 comprise one or more amino acid substitutionsselected from the group consisting of D28(V_(L))Q; D28(V_(L))G;N30(V_(L))S; T31(V_(L))S; A32(V_(L))G; Y49(V_(L))W, Y49(V_(L))D,Y49(V_(L))V; F53(V_(L))W, F53(V_(L))V, F53(V_(L))Q, Y55(V_(L))W,R66(V_(L))N, H91(V_(L))F, H91(V_(L))Y, Y92(V_(L))W, T94(V_(L))S,W95(V_(H))Y, D98(V_(H))W, D98(V_(H))R, D98(V_(H))K, D98(V_(H))H,F100(V_(H))P, F100(V_(H))L, F100(V_(H))M, Y100a(V_(II))F, Y102(V_(II))V,Y102(V_(II))K, and Y102(V_(II)) L.
 50. The antibody of claim 45, whereinthe hypervariable regions of SEQ ID NOs 1 and 2 comprise one or moreamino acid substitutions selected from the group consisting ofY49(V_(I))D, F53(V_(I))W, Y55(V_(I))W, D98(V_(II))W, F100(V_(II))P, andY102(V_(H))L.
 51. The antibody of claim 50 wherein the hypervariableregions of SEQ ID NOs: 1 and 2 comprise the following substitutions:Y49(V_(I))D, F53(V_(I))W, Y55(V_(L))W, F100(V_(H))P, and Y102(V_(H))L.52. The antibody of claim 51 wherein the hypervariable regions of SEQ IDNO 2 further comprise the substitution D98(V_(H))W.
 53. The antibody ofclaim 50 wherein the hypervariable regions of SEQ ID NOs: 1 and 2comprise the following substitutions: Y49(V_(L))D, F53(V_(L))W,Y55(V_(L))W, F100(V_(L))P, and Y102(V_(H))K.
 54. The antibody of claim53 wherein the hypervariable regions of SEQ ID NO 2 further comprise thesubstitution D98(V_(H))W.
 55. The antibody of claim 45, wherein thebinding affinity of the antibody for the HER2 extracellular domain is atleast about three-fold better than the binding affinity of humanizedmonoclonal antibody 4D5-8 for the HER2 extracellular domain.
 56. Theantibody of claim 45, which is a humanized antibody.
 57. The antibody ofclaim 45, which is a human antibody.
 58. The antibody of claim 45, whichis an antibody fragment selected from the group consisting of Fv, Fab,Fab′ and F(ab′)₂ fragments.
 59. An antibody that is capable of bindingto the extracellular domain of HER2 which comprises the light chainvariable domain of SEQ ID NO: 1 wherein one or more amino acids selectedfrom the group consisting of Q27(V_(L)), N30(V_(L)), Y49(V_(L)),F53(V_(L)), Y55(V_(L)), H91(V_(L)), Y92(V_(L)), and T94(V_(L)), numberedaccording to the Kabat numbering system, are substituted with any aminoacid other than alanine.
 60. The antibody of claim 59, wherein the lightchain variable domain of SEQ ID NO: 1 comprises one or more amino acidsubstitutions selected from the group consisting of N30(V_(L))S,Y49(V_(L))F, Y49(V_(L))W, F53(V_(L))W, Y55(V_(L))W, H91(V_(L))F,Y92(V_(L))W, and T94(V_(L))S.
 61. The antibody of claim 60, whereinN30(V_(I)) is substituted with S, H91(V_(I)) is substituted with F, andY92(V_(L)) is substituted with W.
 62. A humanized anti-HER2 antibody4D5-8, comprising one or more amino acid substitutions selected from thegroup consisting of N30(V_(L))S, Y49(V_(L))F, V49(V_(L))W, F53(V_(L))W,Y55(V_(L))W, H91(V_(L))F, Y92(V_(L))W, T94(V_(L))S, F100(V_(L))W,D98(V_(H))W, 100a(V_(H))F, and Y102(V_(H))V, numbered according to theKabat numbering system.
 63. A humanized anti-HER2 antibody 4D5-8 whichcomprises one or more amino acid substitutions selected from the groupconsisting of Y49(V_(L))D, F53(V_(L))W, Y55(V_(L))W, F100(V_(H))P,Y102(V_(H))K, and Y102(V_(H))L.
 64. The antibody of claim 63 furthercomprising the amino acid substitution D98(V_(H))W.
 65. The antibody ofclaim 62 comprising the one or more of the amino acid substitutionsselected from the group consisting of Y49(V_(L))D, F53(V_(L))W, andY55(V_(L))W.
 66. The antibody of claim 62 comprising one or more of theamino acid substitutions selected from the group consisting ofF100(V_(H))P, Y102(V_(H))K, and Y102(V_(H))L.
 67. The antibody of claim64 comprising the following amino acid substitutions: Y49(V_(L))D,F53(V_(L))W, Y55(V_(L))W, F100(V_(H))P, and Y102(V_(H))K.
 68. Theantibody of claim 64 comprising the following amino acid substitutions:Y49(V_(L))D, F53(V_(L))W, Y55(V_(L))W, F100(V_(L))P, and Y102(V_(L))L.69. An article of manufacture comprising a container, a compositioncontained therein, and a package insert or label indicating that thecomposition can be used to treat cancer characterized by theoverexpression of HER2, wherein the composition comprises the antibodyof claim 60 or
 61. 70. The article of manufacture of claim 69, whereinthe cancer is breast cancer.
 71. An antibody variant of a parentantibody which binds HER2, comprising an amino acid substitution atposition 98 of a heavy chain variable domain thereof, and wherein thebinding affinity of the antibody variant for HER2 is better than thebinding affinity of the parent antibody for HER2.
 72. The antibodyvariant of claim 71, wherein the amino acid at position 98 issubstituted with W.
 73. The antibody variant of claim 70, wherein theparent antibody is a humanized antibody.
 74. A method for isolatinghigh-affinity variants of a humanized anti-HER2 antibody, comprising:(a) producing anti-HER2 variants with substitutions at one or more aminoacids selected from the group consisting Q27(V_(I)), D28(V_(I)),N30(V_(I)), T31(V_(I)), A32(V_(I)), Y49(V_(I)), F53(V_(L)), Y55(V_(L)),R66(V_(L)), H91(V_(L)), Y92(V_(L)), T94(V_(L)), W95(V_(H)), D98(V_(H)),F100(V_(H)), Y100a(V_(H)), and Y102(V_(H)), within the hypervariableregions of SEQ ID NOs: 1 and 2, wherein numbering is according to theKabat numbering system; (b) measuring binding affinities of the variantsproduced in (a) for HER2 extracellular domain; and (c) selecting forhigh-affinity variant.